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Category : Protein Folding

Proteasomal degradation of the intrinsically disordered protein tau at single-residue resolution – Science Advances

INTRODUCTION

Intrinsically disordered proteins (IDPs) are abundant in the human proteome and are implicated as therapeutic targets in major human diseases (1). IDPs have amino acid sequences of low complexity and lack an ordered three-dimensional (3D) structure (1). This allows IDPs to dynamically bind to diverse interaction partners and thus influence many biological processes (1). The activity of IDPs is regulated by posttranslational modifications including phosphorylation and truncation (1, 2). Because of their structural instability, IDPs are particularly sensitive to proteolytic degradation (35).

Aggregation of IDPs into insoluble deposits is the hallmark of neurodegenerative diseases (3). Aggregates of the IDP tau are linked to the progression of Alzheimers disease (AD) and are found in other age-related disorders termed tauopathies (6). The longest tau isoform in the human central nervous system comprises 441 residues (7). The N-terminal ~150 residues of tau project away from the microtubule surface and are thus termed projection domain (8). The central part of the tau sequence is formed by pseudo-repeats, which bind to microtubules (8, 9) and are essential for pathogenic aggregation and folding into cross- structure in tau amyloid fibrils (10, 11). Phosphorylated tau accumulates during the development of AD (6, 12).

The 20S proteasome forms the proteolytic core particle of the 26S proteasome holoenzyme (13). In contrast to the proteasomal degradation of most cellular proteins, IDPs can be degraded by the 20S proteasome in an ubiquitin- and adenosine triphosphate (ATP)independent process without the necessity of the 19S regulatory particle (35). Soluble tau is degraded by the 20S proteasome (14, 15), while phosphorylation and aggregation of tau inhibit its turnover by the proteasome (2, 1517). Decline of proteasomal activity and accumulation of tau have been linked to neurodegeneration (2, 18, 19): Decreased proteasomal activity results in tau accumulation, neurotoxicity, and cognitive dysfunction in cell and animal models of neurodegenerative disorders. Pharmacological activation of the 20S proteasome, direct administration of proteasome, or targeted proteasomal degradation of tau is therefore the focus of current therapeutic strategies targeting tauopathies (20, 21).

Here, we study the degradation of the IDP tau by the 20S proteasome through a residue-specific and quantitative approach that combines nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). We provide detailed insights into the identity and properties of the proteasomal degradation products of tau, the single-residue degradation kinetics, and their specific regulation by phosphorylation in different tau domains/by different kinases.

The 20S proteasome (20S) is a barrel-shaped complex comprised by two stacked heptameric -rings that are sandwiched by two heptameric -rings (Fig. 1A) (13). The proteolytic sites, which hydrolyze the peptide bonds of substrates, are located in the subunits. IDPs thus traverse through the -rings to reach the active sites in the interior of the 20S proteasome (Fig. 1B). To study degradation of the IDP tau, we recombinantly prepared 20S from Thermoplasma acidophilum, which contains only one type of subunit and one type of subunit. This 20S particle thus has 14 identical chymotrypsin-like active sites, which are positioned at equal distances around the -rings (Fig. 1B). Electron microscopy (EM) showed intact barrel-shaped 20S complexes (Fig. 1C). The 441-residue isoform of tau (hTau40; also termed 2N4R tau; Fig. 1D) was also expressed in Escherichia coli.

(A) Schematic representation depicting the architecture of the 20S proteasome (20S) comprising 28 subunits arranged in four heptameric rings (7777). (B) The proteolytic active sites of the 20S proteasome are located in its interior, thus enabling degradation of hTau40 into short peptides once it has entered the 20S core. (C) Negatively stained EM micrograph of the 20S proteasome. (D) Domain organization of full-length hTau40 composed of 441 amino acids (aa) (UniProt ID 10636-8). N1 and N2 are the two inserts in the N-terminal projection domain, P1 and P2 correspond to the two proline-rich regions, and R1 to R are five pseudo-repeats. (E) (Left) SDS-PAGE gel showing hTau40 (1) and the degradation of (2 to 5) hTau40 by the 20S proteasome over time. The samples were incubated at 37C for 30 min (2), 90 min (3), and 150 min (4) and were subsequently put at 4C for additional 48 hours (5). After 48 hours, two well-resolved bands at ~28 and ~30 kDa (red lined box) appeared. (Right) The amino acid sequences of the upper (~30 kDa) and lower bands were identified with in-gel analysis and marked in red. Both intermediates correspond to the N-terminal domain of hTau40.

Recombinant hTau40 was incubated with the 20S proteasome, and degradation was followed by SDSpolyacrylamide gel electrophoresis (PAGE) (Fig. 1E, left). After ~150 min, a clear decrease in the intensity of the hTau40 band at ~60 kDa was apparent (lane 4 in Fig. 1E). In addition, two bands running at ~30 and 28 kDa appeared. Analysis after 48 hours of incubation confirmed the presence of the two new bands, while the full-length protein was degraded to near completion (lane 5 in Fig. 1E).

The two intermediate bands were precisely and independently excised from the gel, subjected to in-gel digestion using trypsin, which specifically cleaves at the peptide bond C terminus of lysine or arginine residues, and analyzed using liquid chromatography (LC)MS/MS. For both bands, the MS analysis confidently identified several peptides from the N-terminal domain (Fig. 1E, right). No peptides were identified in the region from 127 to 210, which contains multiple lysine and arginine residues such that trypsin digestion will produce too short sequences to be analyzed by LC-MS/MS. In the case of the upper band, the additional peptide RTPSLPTPPTR (residues 211 to 221 of hTau40) was identified (Fig. 1E, right).

We also separated the two long fragments using LC and detected their molecular weight by intact MS, giving masses of 25.782 and 22.257 kDa (fig. S1). Manual matching of the determined masses to N-terminal sequences of hTau40 showed that the long fragment contains residues 1 to 251, and the short one has residues 1 to 218. Previous studies showed that the upper band is recognized by the antibody Tau-5 (14), which binds to residues in the region from 218 to 225 (22).

To gain insight into the structural properties of the long tau fragments generated during 20S degradation, we recombinantly prepared a tau protein comprising residues 1 to 239 of hTau40. Tau(1239) contains the full epitope for the Tau-5 antibody (residues 218 to 225) and has a length in between the two long N-terminal fragments. Particle size analysis by dynamic light scattering (fig. S2A) showed that both hTau40 and Tau(1239) are more compact than the average size values for IDPs (fig. S2B) (23). hTau40, with an experimental size of 5.2 nm and an expected size for its number of residues of 5.5 nm, is 5% more compact than expected, while Tau(1239) is 18% more compact than expected with 3.3 and 4 nm as experimental and expected sizes, respectively. Despite the stronger compaction of Tau(1239), both proteins present the typical pattern of random coil conformation in circular dichroism spectra (fig. S2C).

Figure S2D shows the 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum of 15N-labeled Tau(1239). The backbone cross peaks are located in the region between 7.6 and 8.6 parts per million (ppm), which is characteristic for IDPs. When compared to hTau40, chemical shift perturbation was restricted to the most C-terminal residues of Tau(1239) (fig. S2E), i.e., residues where Tau(1239), but not hTau40, ends. Analysis of the secondary structure propensities using the chemical shifts of carbonyl and C (fig. S2F) furthermore showed that both hTau40 and Tau(1239) are mainly random coil, in agreement with circular dichroism spectra (fig. S2C).

In addition, the single-residue analysis showed that Tau(1239) contains elements of transient secondary structure: residues 116 to 119 with a tendency for helical structure and two short stretches (residues 150 to 152 and 225 to 230) with extended conformation. The same transiently structured regions were detected in hTau40 (fig. S2F). TALOS+ also identified four regions with preference for extended conformation (residues 275 to 279, 306 to 310, 337 to 339, and 392 to 399) and one with helical content (residues 431 to 437) in hTau40, in agreement with previous analysis (24). The presence of extended conformations in the repeat region has previously been suggested to be responsible for the observation that the repeat region of tau, which is not present in Tau(1239), is less compact when compared to a pure random coil conformation. The combined data thus point to a compaction of the N-terminal cleavage intermediates of hTau40 (fig. S2, A and B).

To identify short tau peptides generated by 20S, we analyzed the released peptides in the supernatant after incubation of hTau40 and 20S using MS. The largest fraction of identified peptides was from hTau40s pseudo-repeat region (Fig. 2, A and B). In addition, peptides from the C-terminal domain and the residue regions 2 to 13, 84 to 103, and 167 to 192 were detected but with very low responses in MS in the supernatant (Fig. 2C). The tau peptides and their cleavage sites identified by MS are generally in good agreement with the proteasomal cleavage sites predicted by NetChop 3.1 (Fig. 2B) (25).

(A) Domain organization of hTau40. (B) Amino acid sequence of hTau40 depicting in color [color code as in (A)] the 20S-generated peptides, which were identified by LC-MS/MS. The peptides underlined with black dots were also present in the in-solution sample but with low intensities. The slashes depict all identified cleavage sites. Cleavage sites predicted by the NetChop server are marked by arrows. The bar on top of the VQIVYK sequence indicates the ability of this sequence to form amyloid-like filaments (26). (C) (Left) Histogram representation of the peak area of 20S-generated tau peptides [color code as in (A)] identified by in-solution analysis. Insert depicting the sequences of the identified peptides and the cleavage sites (marked with slashes). (Right) Histogram representing the most intense peptides in the R3 region. A.U., arbitrary units. (D) ThT fluorescence during incubation of the peptide 309VYKPVDL315. The peptide (50, 100, and 150 M) was incubated with heparin (peptide:heparin molar ratio of 4:1) in triplicates.

The peptide with the highest ion peak area was 309VYKPVDL315 (Fig. 2C, right). It partially overlaps with the hexapeptide sequence 306VQIVYK311 at the beginning of pseudo-repeat R3 (Fig. 2B).

The 306VQIVYK311 sequence is the most hydrophobic residue stretch of tau, is a major driving force for pathogenic tau aggregation, and can form amyloid-like filaments in isolation (26). We therefore tested whether the 20S-generated tau peptide 309VYKPVDL315 can aggregate into amyloid fibrils. To this end, the 309VYKPVDL315 peptide was incubated with heparin at a molar ratio of 4:1.

Figure 2D shows the results from thioflavin-T (ThT) fluorescence measurements of 309VYKPVDL315/heparin samples at three different peptide concentrations during incubation at 37C for 6 days. For all of the samples, the background-corrected ThT intensity was very low and did not increase during incubation (Fig. 2D). No increase in ThT intensity was detected even when the peptide was incubated for 6 days in the absence of heparin (fig. S3). Because ThT fluorescence intensity increases upon binding to amyloid fibrils, the data show that the 20S-generated peptide 309VYKPVDL315 is not able/has a very low propensity to form amyloid fibrils.

To gain insight into the kinetics of degradation of tau by the 20S proteasome and define its residue specificity, we used NMR spectroscopy. Figure 1A displays the 2D 1H-15N HSQC spectrum of 15N-labeled hTau40. The NMR spectrum was recorded at 5C to attenuate the exchange of amide protons with solvent and thus exchange-induced NMR signal broadening. Comparison of the HSQC spectrum of hTau40 alone with the spectra recorded after 30 min and 66 hours (red) in the presence of 20S (hTau40:20S molar ratio of 4:1) showed that after 30 min, the spectrum of hTau40 was essentially unchanged (fig. S4), but after 66 hours, additional sharp cross peaks were present. Four of the newly appearing cross peaks overlapped with signals observed in a natural abundance 1H-15N HSQC spectrum of the 309VYKPVDL315 peptide, i.e., the peptide with the highest ion peak area in MS (fig. S5). The degradation-associated cross peaks were not observed for a separate sample, which additionally contained the proteasome inhibitor oprozomib (Fig. 3A, right spectrum).

(A) Superposition of 2D 1H-15N HSQC spectra of hTau40 at 5C in the presence of the 20S proteasome after 3 hours (black) and 66 hours (red) in the absence (left) and presence (right) of the proteasome inhibitor oprozomib. (B) (Top) Evolution of relative peak intensities, I(t)/I0, in 2D 1H-15N HSQC spectra of hTau40 in the presence of 20S with increasing incubation time at 5C. I0 is the cross-peak intensity observed in the first HSQC. (Middle) Residue-specific rate constants of a first-order model of the 20S degradation kinetics of hTau40. Correlation coefficients for the fit to the first-order model are color-coded (color code bar to the right). Error bars represent SD. (Bottom) Evolution of relative peak intensities in 2D 1H-15N HSQC spectra of hTau40 in the presence of the 20S proteasome and the proteasome inhibitor oprozomib.

When tau is degraded by the proteasome into small peptides, the chemical environment of residues changes. To gain insights into the kinetics of 20S degradation, the intensity of IDP cross peaks at their location in the absence of 20S can be analyzed (27). Because a 1H-15N backbone correlation can be observed for every non-proline residue in the 2D 1H-15N HSQC, up to 397 (441 residues minus the C terminus and 43 prolines, and depending on signal overlap) sequence-specific probes for tau degradation are thus available.

The top panel in Fig. 3B displays the decrease of NMR signal intensities along the hTau40 sequence with increasing 20S incubation time. The fastest decrease occurred in the repeat domain. To derive residue-specific degradation rates, we fitted first-order decay kinetics via linear regression to the residue-specific intensity data. The highest rates occurred in repeat R3 and reached up to 0.015 hours1 at 5C (Fig. 3B, middle, and table S1). Fast degradation kinetics were also observed in the other pseudo-repeats, in agreement with similar sequence compositions. In addition, taus C terminus as well as residues ~220 to 250 at the end of the proline-rich region were rapidly affected by degradation.

Oprozomib predominantly inhibits the chymotrypsin-like activity of the 20S proteasome (28). Detailed analysis of the hTau40 spectra in the presence of both 20S and the small-molecule oprozomib showed that the cross peaks of residues in R2 and R3 decreased in intensity by up to 20% after 66 hours (Fig. 3B, bottom, and table S1). Thus, the 20S complex has residual proteolytic activity, which is not inhibited by oprozomib.

A large number of kinases can phosphorylate tau (29). These include proline-directed kinases [e.g., glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (cdk5)] that phosphorylate proline-serine/threonine motifs, notably in the proline-rich region of tau, as well as non-prolinedirected kinases [e.g., microtubule affinity-regulating kinase (MARK), protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII)], which phosphorylate the KXGS motifs in the pseudo-repeats. CaMKII phosphorylates tau at several sites (30) and colocalizes with neurofibrillary tangles (NFTs) in AD brains (31).

To gain insight into the influence of substrate phosphorylation on 20S degradation, we phosphorylated recombinant hTau40 with CaMKII in vitro. SDS-PAGE demonstrated an upfield shift in the hTau40 band, confirming successful phosphorylation (Fig. 4A). According to MS/MS analysis, CaMKII phosphorylates S131 and T135 in the projection domain, T212 and S214 in P2, S262 in R1, and S356 in R4 (30). 1H-15N NMR spectroscopy further showed that S214, S356, and S413 are fully phosphorylated in hTau40 (Fig. 4B). In addition, S262, S324, and S352 were found to be partially phosphorylated (Fig. 4B).

(A) SDS-PAGE gel demonstrating phosphorylation of hTau40 by CaMKII in the presence of calmodulin. (B) (Left) Enlarged region with phosphorylated residues taken from the first 2D 1H-15N HSQC recorded at 5C for a total duration of 3 hours on CaMKII-phosphorylated hTau40 in the presence of 20S. On top, the location of the phosphorylated residues is marked by short black lines in the context of the domain diagram of hTau40. (Right) Superposition of the first 2D 1H-15N HSQC spectrum (black; total measurement time: 3 hours) of CaMKII-phosphorylated hTau40 in the presence of 20S with the spectrum completed after 66 hours (red). (C) Relative peak intensities in 2D 1H-15N HSQC spectra of CaMKII-phosphorylated hTau40 in the presence of the 20S proteasome with increasing time of incubation at 5C (from red to blue).

We then incubated CaMKII-phosphorylated hTau40 with 20S proteasome at 5C. Even after 66 hours, no degradation peaks were observed in the 1H-15N HSQC spectrum (Fig. 4B and fig. S6). In addition, hTau40 cross-peak intensities remained largely unaffected (Fig. 4C and fig. S6). Similarly, CaMKII phosphorylation of the tau construct K18, which only contains the repeat domain, attenuated its degradation by the 20S proteasome (fig. S7). Thus, phosphorylation of tau by CaMKII interferes with the degradation of tau by the 20S proteasome.

GSK3 is ubiquitously expressed in mammalian tissue and has been implicated as a major tau kinase in AD (32). In vitro modification of hTau40 by GSK3 results in phosphorylation of S46, T175, T181, S202, T205, T212, T217, T231, S235, S396, S400, and S404 (33). NMR confirmed complete phosphorylation of S396, S400, and S404 (Fig. 5A). In contrast to CaMKII phosphorylation (Fig. 4), phosphorylation by GSK3 did not block proteasomal processing of hTau40 [Figs. 5A (red spectrum) and 6]. Analysis of cross-peak intensities at increasing 20S incubation times further showed that rapid degradation occurred in repeats R2 and R3 of hTau40 (Fig. 5B).

(A) (Left) Enlarged region with phosphorylated residues taken from the first 2D 1H-15N HSQC recorded at 5C for a total duration of 3 hours on GSK3-phosphorylated hTau40 in the presence of 20S. On top, the cartoon depicts the sites of phosphorylation of hTau40 by GSK3. (Right) Superposition of the first 2D 1H-15N HSQC spectrum (black; total measurement time: 3 hours) of GSK3-phosphorylated hTau40 in the presence of 20S with the spectrum completed after 66 hours (red). (B) Relative peak intensities in 2D 1H-15N HSQC spectra of GSK3-phosphorylated hTau40 in the presence of the 20S proteasome with increasing time of incubation at 5C (from red to blue).

(A and B) Per-residue rate constants for degradation of tau by the 20S proteasome. Residue-specific rate constants of a first-order model of the 20S degradation kinetics of hTau40 at 5C (A, top; same as in Fig. 3B), in the presence of the inhibitor oprozomib (A, bottom), of hTau40 phosphorylated by CaMKII (B, top), and of hTau40 phosphorylated by GSK3 (B, bottom). Correlation coefficients for the fit to the first-order model are color-coded (color code bars to the right). Error bars represent SD. (C) Schematic representation illustrating the phosphorylation-dependent degradation of the AD-related protein tau by the 20S proteasome: Wild-type tau (hTau40) is degraded by the 20S proteasome starting from the pseudo-repeat region and the C-terminal domain, producing short peptides (blue, pink, and orange) from those regions, followed by degradation of the N-terminal domain, which generates two long N-terminal fragments. Depending on the sites of phosphorylation, 20S degradation of tau is inhibited (CaMKII; top) or attenuated (GSK3; bottom). The color code of different hTau40 domains is described in Fig. 1.

Figure 6 (A and B) compares the residue-specific degradation rates of unmodified hTau40 in the presence of the 20S proteasome (Fig. 6A, top), unmodified hTau40 in the presence of 20S and the inhibitor oprozomib (Fig. 6A, bottom), CaMKII-phosphorylated hTau40 and 20S (Fig. 6B, top), and GSK3-phosphorylated hTau40 and 20S (Fig. 6B, bottom, and table S1). As calculated from the time-dependent decrease in cross-peak intensities, GSK3-phosphorylated hTau40 is most efficiently processed by the 20S proteasome in repeats R2 and R3. The phosphorylation of selected residues in taus C-terminal domain, however, blocks cleavage of peptide bonds in this region. In addition, the decay of NMR signals in the proline-rich region was strongly attenuated (Fig. 5B and fig. S4), in agreement with phosphorylation of T212, T217, T231, and S235 by GSK3 (33).

Within the cell, IDPs are constantly synthesized and degraded by the proteasome. Because they lack a globular structure, IDPs can directly be processed by the 20S proteasome without the need for previous ubiquitination and unfolding by the 26S proteasome (35, 34). In parallel, IDPs can be degraded in a ubiquitin-dependent manner by the 26S proteasome. Aggregates of IDPs cannot properly be degraded by the proteasome and are instead processed through autophagy (18, 19). In addition, tau aggregates might inhibit the activity of proteasomes and thereby contribute to neurodegeneration (2, 17, 18). Detailed insights into the processing of tau and other IDPs by the 20S proteasome may therefore be important for treating neurodegeneration and other human diseases (34).

Inhibition of the proteasome by small molecules results in increased amounts of tau in SH-SY5Y cells and rat brain (14, 35). In addition, the four-repeat isoform hTau43 (also termed 0NR4 tau) was shown to be degraded by the human 20S proteasome in vitro without previous ubiquitination (14). In agreement with the latter study, which used human 20S (14), we observed two relatively stable populations of long tau fragments from the N terminus when incubating hTau40 with the 20S proteasome from T. acidophilum (Fig. 1). To determine the identity of the two hTau40 fragments, we performed MS analysis and found that the long and short fragments contain residues 1 to 251 and 1 to 218, respectively (Fig. 1).

Proteasomes cleave their substrates to short peptides with mean lengths between 6 and 10 amino acids (4, 36). Longer (>50 amino acids) degradation intermediates are rarely detected, because the substrate is thought not to dissociate from the proteasome during the degradation process. The presence of two long truncated tau fragments during 20S degradation is therefore unexpected. The more than 200-residue-long tau fragments contain multiple, potential proteasomal cleavage sites (Fig. 2B). To investigate whether the generation of these fragments is the result of specific structural properties of the N-terminal domain of hTau40, we characterized this domain at a single-residue level by NMR spectroscopy. The analysis showed that Tau(1239) is more compact than hTau40 (fig. S2). We speculate that the more compact structure might interfere with 20S cleavage of the N-terminal fragments.

The short ~6- to 10-residue tau peptides generated by the 20S proteasome can further be cleaved by other proteases (2). In parallel, they might itself contain activity, which is relevant for pathological processes. Consistent with this hypothesis, the six-residue tau peptide 306VQIVYK311 can form insoluble amyloid-like filaments in vitro (26). We therefore used MS to identify the tau peptides generated by 20S degradation (Fig. 2). From the large number of different 20S-generated peptides, the tau peptide with the highest ion peak area was 309VYKPVDL315. Consistent with the high abundance of the 309VYKPVDL315 peptide generated by 20S degradation, signals corresponding to this peptide were identified in the NMR spectra of degraded tau (fig. S4). The 309VYKPVDL315 peptide lacks the first three amino acids of the filament-forming 306VQIVYK311 sequence but has four additional N-terminal residues including the two hydrophobic residues V313 and L315. Despite an overall high hydrophobicity, however, the tau peptide 309VYKPVDL315 did not aggregate into amyloid-like filaments in the presence of the aggregation enhancer heparin (Fig. 2D). Notably, all of the other 20S-generated peptides in the region from 308 to 320 also contain residue P312, i.e., a proline with known -strandbreaking property (Fig. 2C, right). Cleavage of tau by the 20S proteasome thus generates peptides that are unable to aggregate into amyloid-like filaments.

A wide range of assays have been developed to follow protein degradation. These assays often sample the degradation reaction at discrete time points using SDS-PAGE and antibody binding, autoradiography, protein staining, or Western blotting (37). In addition, proteasome activity can be analyzed through the measurement of fluorescence anisotropy of small-molecule dyes attached to substrate proteins. The identity of degradation products can furthermore be determined using MS. Here, we combined MS with NMR to (i) gain insight into the structural properties of the long degradation intermediates of tau identified by MS and (ii) quantify degradation kinetics in the IDP tau with single-residue and high temporal resolution. MS and NMR spectroscopy are thereby complementary, because MS enables large-scale identification of substrate fragments and peptides generated by proteasomal degradation, but cannot identify all released peptides, lacks single-residue resolution, and is limited in temporal resolution. NMR spectroscopy makes it possible to follow substrate degradation, while the reaction occurs in the test tube, and quantify degradation kinetics at high spatial/per-residue and temporal resolution. On the other hand, a high number of generated peptides and fragments complicate their identification by NMR especially for large IDPs, such as tau, which have many cross peaks. In addition, it has to be taken into account that the cleavage of a peptide bond can be sensed by residues that are several positions removed from the site of proteolysis (27). Because of the abovementioned aspects, we believe that the combination of MS and NMR will also be useful to investigate differences in the degradation pattern and substrate selectivity of 20S proteasomes from different organisms.

Using NMR spectroscopy, we found that the 20S degradation of many tau residues follows first-order decay kinetics (Fig. 3). The maximum degradation rate reached ~0.015 hours1 at 5C, which corresponds to a degradation half-time of ~46 hours. The reported half-life of tau in HT22 cells is 60 hours (15). The analysis further showed that the 20S proteasome from T. acidophilum preferentially cleaves tau in the pseudo-repeat region, with the fastest rates observed in repeat R3 (Fig. 3). Repeat R3 is part of the cross- structure of heparin-induced tau fibrils (38). In addition, R3 is located in the core of paired helical filaments purified from the brains of patients with AD (10). The data suggest that the 20S proteasome preferentially degrades the regions of tau, which are important for pathogenic aggregation.

SDS-PAGE analysis, in combination with antibody binding, was used to suggest that the degradation of tau by the 20S proteasome is bidirectional (14), supporting degradation models in which 20S degradation has a preference for the free NH2 or COOH terminus of a substrate (39). In contrast, we find that the proteasome degradation of tau is most efficient in the repeat domain (followed by the C-terminal domain; Fig. 3). Our results are thus in agreement with reports showing that the 20S proteasome can initiate endoproteolytic cleavage at internal sites of IDPs (5). The efficient cleavage of the pseudo-repeat region also enables the generation of the two long fragments from the N terminus of tau (Fig. 1).

The strength of the quantitative, combined MS/NMR approach was further supported by the experiments, in which we studied the influence of phosphorylation of tau on its degradation by the 20S proteasome (Figs. 4 and 5). Tau molecules found in NFTs in the brains of patients with AD are hyperphosphorylated, and dysregulation of tau phosphorylation has been linked to neuronal toxicity (6). Consistent with the hypothesis that impaired proteasomal degradation results in tau accumulation, phosphomimetic tau variants were less efficiently degraded by the proteasome in autophagy-deficient mouse embryonic fibroblasts (16).

The quantitative NMR-based degradation analysis showed that phosphorylation of tau by the non-prolinedirected serine/threonine kinase CaMKII inhibits degradation of tau by the 20S proteasome (Figs. 4 and 6). When the proteasome cannot degrade tau, autophagy becomes important, in agreement with the observation that autophagy is the primary route for clearing phosphorylated tau in neurons (16). However, using the same quantitative approach, we found that tau phosphorylated by GSK3, which phosphorylates Pro-Ser/Thr epitopes seen in NFTs in AD (32), only blocks cleavage in certain regions but does not interfere with tau cleavage in the pseudo-repeats R2 and R3 (Figs. 5 and 6). The regions of tau, which are no longer cleaved such as the C-terminal domain and the proline-rich domain, contain residues phosphorylated by GSK3 (Fig. 5). While GSK3 does not phosphorylate residues in the repeat region, CaMKII phosphorylates S262, S324, S352, and S356 and blocks degradation by the 20S proteasome (Figs. 4 and 6). Phosphorylation of S262, S324, S352, and S356 therefore appears to play an important role in the inhibition of tau degradation by the 20S proteasome. S262, S324, S352, and S356 are also phosphorylated by microtubule-associated protein/MARKs, and their phosphorylation affects tau aggregation as well as microtubule binding of tau (40). Currently, the mechanism of impaired degradation of CaMKII-phosphorylated tau is unknown but could involve (i) an impaired/restricted entry through the 20S gate formed by the first 12 amino acids of the subunit and (ii) a blocked interaction with the catalytic sites in the subunit. Our study provides the basis to quantify with single-residue resolution the degradation of tau and other IDPs, their different isoforms, and posttranslationally modified variants and thus gain mechanistic insight into disease-associated accumulation of IDPs.

Unlabeled and 15N-labeled Tau protein (hTau40, UniProt ID 10636-8, 441 residues) were expressed in E. coli strain BL21(DE3) from a pNG2 vector (a derivative of pET-3a, Merck-Novagen, Darmstadt) in the presence of an antibiotic. In case of unlabeled protein, cells were grown in 1 to 10 liters of LB and induced with 0.5 mM IPTG (isopropyl--d-thiogalactopyranoside) at OD600 (optical density at 600 nm) of 0.6 to 0.8. To obtain 15N-labeled protein, cells were grown in LB until an OD600 of 0.6 to 0.8 was reached, then centrifuged at low speed, washed with M9 salts (Na2HPO4, KH2PO4, and NaCl), and resuspended in minimal medium M9 supplemented with 15NH4Cl as the only nitrogen source and induced with 0.5 mM IPTG. After induction, the bacterial cells were harvested by centrifugation, and the cell pellets were resuspended in lysis buffer [20 mM MES (pH 6.8), 1 mM EGTA, and 2 mM dithiothreitol (DTT)] complemented with protease inhibitor mixture, 0.2 mM MgCl2, lysozyme, and deoxyribonuclease (DNase) I. Subsequently, cells were disrupted with a French pressure cell press (in ice-cold conditions to avoid protein degradation). In the next step, NaCl was added to a final concentration of 500 mM and boiled for 20 min. Denaturated proteins were removed by ultracentrifugation at 4C. The supernatant was dialyzed overnight at 4C against dialysis buffer [20 mM MES (pH 6.8), 1 mM EDTA, 2 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 50 mM NaCl] to remove salt. The following day, the sample was filtered and applied onto a previously equilibrated ion-exchange chromatography column, and the weakly bound proteins were washed out with buffer A (same as the dialysis buffer). Tau protein was eluted with a linear gradient of 60% final concentration of buffer B [20 mM MES (pH 6.8), 1 M NaCl, 1 mM EDTA, 2 mM DTT, and 0.1 mM PMSF]. Protein samples were concentrated by ultrafiltration (5 kDa Vivaspin from Sartorius) and purified by gel filtration chromatography. Last, the protein was dialyzed against 50 mM sodium phosphate (NaP) (pH 6.8).

20S proteasomes from T. acidophilum were expressed from pRSETA containing the bicistronic gene including psmA and psmB. Transformed BL21 cells were induced with 0.1 mM IPTG and incubated for 18 hours at 37C. Harvested cells were resuspended in 3 ml of lysis buffer (50 mM Na2HPO4 pH 8.0, 300 mM NaCl) per 1 g of cells and lysed with the French press. The lysate was incubated at 65C for 15 min. Heat-denatured proteins were removed by centrifugation at 30,000g at 4C. Polyethylenimine (0.1%, w/v) was added to the supernatant to precipitate contaminating nucleic acids. Precipitated nucleic acids were removed by centrifugation at 100,000g for 1 hour. The supernatant was subjected to differential precipitation with polyethylene glycol 400 (PEG; number signifies the mean molecular weight of the PEG polymer). PEG400 was added to a concentration of 20% (v/v) to the supernatant under stirring at 18C and incubated for 30 min. Precipitated proteins were removed by centrifugation at 30,000g for 30 min at 4C. The supernatant was then precipitated by raising the concentration of PEG400 to 40% (v/v). The precipitate of this step contained the 20S proteasomes and was recovered by centrifugation at 30,000g for 30 min at 4C and resuspended in purification buffer (0.05 M BisTris pH 6.5, 0.05 M K(OAc), 0.01 M Mg(OAc)2, 0.01 M -Glycerophosphate) containing 5% (w/v) sucrose, 10 mM DTT, and 0.01% (w/v) lauryl maltose neopentyl glycol (LMNG) on an orbital shaker at 18C. The resuspended material was loaded on 10 to 30% (w/v) sucrose gradients in purification buffer containing 5 mM DTT, which are centrifuged at 284,000g for 16 hours at 4C. Gradients were harvested in 400 l of fractions. SDS-PAGE was used to identify fractions containing 20S proteasomes. Selected fractions were pooled and precipitated by the addition of 40% (v/v) PEG400. After centrifugation (30,000g, 20 min), the supernatant was removed and the precipitate was resuspended in purification buffer containing 5% (w/v) sucrose, 10 mM DTT, and 0.01% (w/v) LMNG. The resuspended material was loaded on linear 10 to 40% (w/v) sucrose gradients in purification buffer containing 5 mM DTT, which are centrifuged at 284,000g for 18 hours at 4C. Fractions containing 20S proteasomes are yet again identified by SDS-PAGE, precipitated and concentrated by the addition of 40% PEG400, and resuspended in purification buffer containing 5% (w/v) sucrose and 5 mM DTT, yielding the final purified protein preparation at 26 mg/ml. Protein concentrations were determined by the Bradford assay (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA) as a standard.

For grid preparation, a protein stock solution (6 mg/ml) was diluted to 0.25 mg/ml with standard buffer without sucrose. Glutaraldehyde was added to the diluted protein solution to a concentration of 0.1% (v/v). After incubation for 2.5 min at room temperature, the reaction was quenched by the addition of 50 mM l-aspartate (pH 6.5). A continuous carbon foil was floated on the protein solution for 1 min at 4C. A holey carbon copper grid was used to remove the continuous carbon foil from the protein solution. Excess liquid was removed with a tissue paper. Proteins were stained by floating the grid on a saturated uranyl formate solution for 1 min at 4C. Remaining staining solution was removed with a tissue, and the grid was dried under ambient conditions. Negative-stain EM images were taken with a Philips CM200 microscope (160 kV). Images were acquired at a magnification of 66,000. The pixel size corresponds to 3.34 per pixel. The TVIPS charge-coupled device camera was used to record the micrographs.

hTau40 was phosphorylated by CaMKII (recombinant human CaMKII alpha protein from Abcam) and GSK3 [recombinant human GSK3 beta protein (active) from Abcam]. The reaction was performed by mixing 0.2 mM hTau40 with 0.02 mg/ml kinase, 2 mM DTT, 2 mM ATP, 1 mM PMSF, and 5 mM MgCl2 in 40 mM Hepes (pH 7.4). In case of CaMKII, we additionally used 2 M calmodulin (bovine calmodulin, recombinant from Sigma), 1 mM CaCl2, and, in case of GSK3, 2 mM EGTA. The samples were incubated at 30C overnight and buffer-exchanged to 50 mM NaP (pH 6.8). Protein concentrations were determined by the Bradford assay using BSA as a standard.

For detection of hTau40 degradation products/fragments generated by the 20S proteasome (hTau40:20S molar ratio of 3:1), we used a 18% separating gel [ddH2O, 30% acrylamide, 1.5 M tris (pH 8.8), 10% SDS, 10% ammonium persulfate (APS), and tetramethylethylenediamine (TEMED)] and a 4% stacking gel [ddH2O, 30% acrylamide, 1 M tris (pH 6.8), 10% SDS, 10% APS, and TEMED]. For validation of hTau40 phosphorylation, we used a 12% separating gel and a 4% stacking gel.

hTau40 was incubated with 20S proteasome for 150 min at 37C and 1 day at 4C. The resulting reaction sample was in 50 mM NaP (pH 6.8). The buffer was exchanged to MS compatible sample buffer using Amicon Ultra centrifugal filters with a molecular weight cutoff of 3000. The filter was first washed using water. The reaction sample and 300 l of sample buffer [0.1% formic acid (FA)] were then added to the filter and centrifuged at 7500g for 30 min. After removing the buffer, 300 l of sample buffer was added and centrifuged for 30 min. The buffer exchange was then repeated one more time. Last, the samples were diluted to 100 ng/l for the following MS analysis.

The intact MS experiment was performed on Q Exactive HF-X2 (Thermo Fisher Scientific) coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific) equipped with a PepSwift Monolithic Trap Column [200 m inside diameter (ID) 5 mm] and a ProSwift RP-4H Monolithic Nano Column (100 m ID 25 cm). The flow rate was set to 1 l/min. Mobile phase A and mobile phase B were 0.1% (v/v) FA and 80% (v/v) acetonitrile (ACN), 0.08% FA, respectively. The gradient started at 20% B and increased to 50% B in 33 min and then kept B constant at 90% for 4 min, followed by re-equilibration of the column with 5% B. MS spectra were acquired with the following settings: microscans, 1; resolution, 120,000; mass analyzer, Orbitrap; automatic gain control (AGC) target, 3 106; injection time, 100 ms; mass range, 450 to 2000 mass/charge ratio (m/z).

hTau40 samples were incubated with 20S proteasome (molar ratio of 3:1) for different times (30, 90, and 150 min at 37C and, additionally, 48 hours at 4C). The samples were then analyzed by SDS-PAGE electrophoresis as described above. The two fragments (around 25 to 30 kDa) were carefully cut from the gel and used for in-gel analysis. The in-gel digestion of the two bands was performed using trypsin (Promega) to the gels. In the next step, the extracted peptides were desalted by using stage tips. In the last step, the samples were dried (SpeedVac) and readied for further analysis.

hTau40 was incubated with 20S proteasome (molar ratio of 3:1) at 37C for 3 hours before the analysis. The samples were precipitated by acetone and put at 30C overnight. Then, the samples were centrifuged at 14,000g for 10 min, and the supernatant was collected and dried. In the next step, contaminates were removed by the sp3 method, followed by direct injection into the mass spectrometer.

In-geldigested peptides were analyzed using an Orbitrap Fusion Tribrid (Thermo Fisher Scientific) instrument. In-solution samples were analyzed using Orbitrap Fusion Lumos (Thermo Fisher Scientific). Both instruments are coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific) equipped with an in-housepacked C18 column (ReproSil-Pur 120 C18-AQ, 1.9 m pore size, 75 m inner diameter, 30 cm length, Dr. Maisch GmbH). Both Orbitrap Fusions (Tribrid and Lumos) were operating in data-dependent mode for MS2. Dried samples were resuspended in 5% ACN, 0.1% FA. Samples were centrifuged for 10 min at 14,000g, and the supernatants were transferred to new sample tubes. In both cases, the flow rate was set to 300 nl/min. Mobile phase A and mobile phase B were 0.1% FA (v/v) and 80% ACN, 0.08% FA (v/v), respectively. The gradient in Orbitrap Fusion Tribrid (in-gel samples) started at 10% B and increased to 42% B in 43 min and then kept B constant at 90% for 6 min, followed by re-equilibration of the column with 5% B. MS1 spectra were acquired with the following settings: resolution, 120,000; mass analyzer, Orbitrap; mass range, 380 to 1500 m/z; injection time, 50 ms; AGC target, 4 105; S-Lens radio frequency (RF) levels, 60; charge state, +2 to +7; dynamic exclusion after n time, n = 1, dynamic exclusion duration = 60 s. MS2 parameters were as follows: first mass, 120; activation type, higher-energy collisional dissociation (HCD); collision energy, 35; Orbitrap resolution, 30,000; maximum injection time, 250 ms; AGC target, 100,000. The gradient in Orbitrap Fusion Lumos (in-solution samples) increased to 30% B in 42 min and further to 40% B in 4 min and then kept B constant at 90% for 6 min, followed by re-equilibration of the column with 5% B. MS1 spectra were acquired with the following settings: resolution, 120,000; mass analyzer, Orbitrap; mass range, 350 to 1600 m/z; injection time, 50 ms; AGC target, 5 105; S-Lens RF levels, 30; charge state, +2 to +7; dynamic exclusion after n time, n = 1, dynamic exclusion duration = 30 s. MS2 parameters were as follows: first mass, 120; activation type, HCD; collision energy, 30; Orbitrap resolution, 15,000; maximum injection time, 120 ms; AGC target, 100,000.

Thermo Proteome Discoverer (2.1.0.81) was used for database searching. In Proteome Discoverer, the Sequest HT, fixed value peptide spectrum match validator, and Precursor Ions Area Detector nodes were used. Parameters for database searching were as follows: the hTau40 protein sequence (P10636-8) was downloaded from Swiss-Prot. Mass tolerance for precursors and fragment ions was set as 10 and 20 ppm, respectively. Maximal missed cleavage was 4. Dynamic modifications were set as oxidation (M) and acetylation (protein N terminus). For in-gel samples, fixed modification was carbamidomethylation (C). Trypsin was used as the enzyme, and its specificity was set as semi-specific. For in-solution sample, no enzyme was set. For precursor ions area detector, mass precision was 2 ppm. Only the peptides that were identified with high confidence were used in this study. For in-solution samples, the peak area of precursors was used for quantification of the identified peptides.

The peptide VYKPVDL was synthesized as trifluoroacetic acid salts by GenScript, and the stock solution (1 mM) was made in 25 mM Hepes (pH 7.4). To test whether the peptide can aggregate into amyloid fibrils, we used 50, 100, and 150 M of the peptide in 25 mM Hepes (pH 7.4). The stock solution of ThT (purchased from Sigma) was prepared in ddH2O, and for the binding assay, 50 M was used. When heparin (~20 kDa, Roth) was added to the sample, the molar ratio of the peptide to heparin was 4:1. ThT fluorescence was then measured with excitation at 440 nm and emission at 482 nm at 37C using a multimode microplate reader (Spark 20M, TECAN).

2D 1H-15N HSQC and 3D spectra (HNCO and HNCA) of hTau40 and Tau(1239) were acquired at 5C on a Bruker 800 MHz spectrometer equipped with triple-resonance 5-mm cryogenic probe. The protein concentration was 125 M in 50 mM NaP buffer (pH 6.8), 5% D2O, 0.1% NaN3, and 50 M dextran sulfate sodium. Spectra were processed with TopSpin 3.5 (Bruker) and analyzed using Sparky.

NMR degradation experiments with 20S proteasome involving hTau40, phosphorylated hTau40, and hTau40 in the presence of the proteasome inhibitor were acquired and processed as explained above. 2D 1H-15N HSQC spectra were recorded for 15N-labeled hTau40 and 20S proteasome in a molar ratio of 4:1 in 50 mM NaP buffer (pH 6.8) and 10% D2O. The dead time between mixing hTau40 and 20S proteasome and starting the first HSQC experiments was ~30 min.

To study the kinetics of the degradation of hTau40 by the 20S proteasome, 60-min HSQCs were measured every hour during the first 24 hours and then for 180-min HSQCs every 3 hours (for a total of 38 measurements) up to 66 hours. In case of the sample with the inhibitor as well as the phosphorylated samples, 180-min HSQCs were recorded every 3 hours for a total of 22 measurements (66 hours). For our control sample, we used the proteasome inhibitor oprozomib (ApexBio), which was incubated for 2 hours at 37C in 250 molar excess before the experiment.

Peak intensities were extracted from a series of 1H-15N HSQC datasets at predetermined time intervals. After peak assignment with the software Sparky, the peak intensities were normalized with respect to the initial peak intensity for each residue, taking into account the duration of each HSQC. A residue was excluded from plotting and further analysis if a consecutively recorded peak intensity increased to more than 115% of the relative intensity of the preceding measurement. Such an increase in peak intensity when compared to the preceding measurement can arise from more favorable relaxation properties in the generated peptides when compared to full-length tau. In addition, peak overlap can potentially cause fluctuating intensities.

The peak intensities at all recorded times of the remaining (i.e., not excluded) residues were analyzed by fitting to first-order decay kinetics via linear regression of the data with respect to the analytic solution of the normalized first-order decay model. The fitted first-order decay reaction constants were plotted for all nonexcluded residues of hTau40. The statistical uncertainty in the determined degradation rates expressed in terms of SDs of fits was estimated as follows. For each sample, we randomly excluded five (in case of samples hTau40 + 20S in molar ratios of 4:1 and 4.5:1) or three (in case of samples hTau40 + 20S + inhibitor, hTau40 + CaMKII + 20S, and hTau40 + 20S + GSK3) intensity profiles collected at the various time intervals from the fitting procedure and repeated this procedure 20 times. The selection was performed by randomly drawing five (three, respectively) numbers from a uniform distribution over all profiles measured at different time intervals, and the fitting procedure was carried out on each of these subsamples and each amino acid residue. From the 21 fits per residue obtained this way (20 undersampled plus 1 fit based on all measured profiles), we calculated the sample SD and depicted it as error bars. The plots depicting degradation rates were plotted as the full-data fit (declared here as the mean estimated value) plus/minus the SD. In addition, we determined the Pearson correlation coefficients for all respective fits, which are encoded in the color. Fits with an incorrect sign of (i.e., implying an incorrect/unphysical trend) were excluded from the plot.

Acknowledgments: We thank N. Rezaei-Ghaleh for help with NMR experiments and the Max Planck society for support. Funding: The financial support from the German Research Foundation (DFG) through the Emmy Noether Program GO 2762/1-1 (to A.G.) is acknowledged. P.F. is supported by a Manfred-Eigen-Fellowship from the Max Planck Institute for Biophysical Chemistry. M.Z. was supported by the advanced grant 787679-LLPS-NMR of the European Research Council. Author contributions: T.U.-G. performed tau phosphorylation, NMR experiments, and data analysis. P.F. and K.-T.P. performed MS and data analysis. A.I.d.O. analyzed Tau(1239) and performed NMR experiments and K18 degradation. F.H. prepared 20S proteasome. A.G. performed NMR data analysis. M.-S.C.-O. prepared Tau(1239). A.C. supervised 20S preparation. H.U. supervised MS. E.M. and M.Z. designed the study. The manuscript was written through contributions of all authors. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015349. The chemical shifts of Tau(1239) were deposited in the BMRB (identifier: 28065). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Proteasomal degradation of the intrinsically disordered protein tau at single-residue resolution - Science Advances

Recommendation and review posted by Alexandra Lee Anderson

Solve Puzzles for Science | Foldit

(This post was originally sent out on July 3 to our mailing list. You can sign up for the mailing list here to receive weekly updates about Foldit, including tips and tricks and see the top-scoring solutions to the week's puzzles. Don't forget to join our Discord as well to stay in the chat even when you're not folding!)

Hey folders!

Dev Josh here with your weekly Foldit update.

This week we saw the introduction of the Reaction Design tool. The devs are working hard on polishing it up and making it more usable! As always, thanks for your feedback and bug reports. You can submit more feedback here.

In this puzzle, I accidentally evo'ed on a broken developer build and got the top score. Whoops, sorry about that!Here are some of the solutions at the top of the leaderboards. [A note from our scientists: the top of the leaderboards doesn't always mean the most scientifically useful. These highlights are not scientific feedback and are not officially endorsed as scientifically valid designs by the Foldit team.]

Join the mailing list to see what others are folding!

This week's recipe is an oldie but a goodie from drjr. The recipe is called Reset, and it does what it says on the tin: reset to the best score, unfreeze the protein, remove all your bands, and set the CI to 1. A simple recipe, but a handy quality of life tool for when you just need to backtrack a little.

Quick shoutout to argyrw for always being a friendly voice in chat! Say hi to her in global or veteran chat.

Beginner: Are you still using Pull to draft your protein in the early game? Try making cutpoints and moving pieces around with the Move tool, it's so much easier! Don't forget to disable cutpoint bands in the Behavior tab, or they'll all come together again when you wiggle.

Intermediate: It can be really tempting mid-game to just switch to running recipes. But give some time to carefully inspect every acceptor and donor (the red and blue dots) to see what hydrogen bonds you can form, and manually mutate as needed. Not only will this lower your BUNS, but it'll help form a strong hbond network. The scientists love this, and your rank will too!

Expert: If you haven't already, read bkoep's blog on binder design metrics. DDG, SASA, and SC are going to become really important soon since we're looking to add objectives for them. So understanding and practicing these principles now can help you get a headstart on the competition! Use the protein design sandbox to try out some ideas.

Have a tip to share or a recipe to recommend? Reply with your suggestions or make a wiki page for your ideas! Reaction Design doesn't have a page yet, so if you understand this tool, help out your community by writing about it! (Since writing this post, LociOiling has graciously created the page for Reaction Design puzzles.)

Until next time, happy folding!

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Solve Puzzles for Science | Foldit

Recommendation and review posted by Alexandra Lee Anderson

Setting the bar in education – The Star Online

Cheahs belief in working with the best and learning from the best also birthed the appointments of the Jeffrey Cheah distinguished professors.

Under the collaboration between Jeffrey Cheah Foundation and globally acclaimed academic institutions, eminent experts and scholars - who have contributed to solving critical global issues in health, disease and economy amongst others - are appointed to share their knowledge and expertise with Malaysian academics, students and the general public.

Among the prominent names on the list are:

Prof Jeffrey David Sachs

As a world renowned economist and director of the UN Sustainable Development Solutions Network, Prof Sachs is one of the worlds most influential experts on sustainable economic development.

A passionate leader in the fight against poverty and the special advisor to the UN secretary-general on sustainable development, he has advised heads of states and governments on economic strategy for more than a quarter century.

Appointed as an honorary Jeffrey Cheah distinguished professor of sustainable development at Sunway University this year, he is also the chairman of the Jeffrey Sachs Centre on Sustainable Development.

Prof Sir Leszek Borysiewicz

The chairman of Cancer Research United Kingdom (UK) since 2016, Prof Borysiewicz is an Honorary Jeffrey Cheah distinguished professor who is now the emeritus vice-chancellor of the University of Cambridge, after serving as its vice-chancellor from 2011 to 2017.

A founding fellow of the Academy of Medical Sciences, he has been chief executive of the UKs Medical Research Council since 2007 and was knighted in 2001 for his breakthroughs in vaccines, including developing Europes first trial of a vaccine to treat cervical cancer.

Prof Sir Alan Fersht

World leading protein scientist Prof Fersht, also an honorary Jeffrey Cheah distinguished professor and life fellow of Gonville and Caius College Cambridge, is widely regarded as one of the main pioneers of protein engineering, which is a process to analyse the structure, activity and folding of proteins.

His current research involves a fusion of protein engineering, structural biology, biophysics and chemistry to study the structure, activity, stability and folding of proteins, as well as the role of protein misfolding and instability in cancer and disease.

Prof Kay-Tee Khaw

Prof Khaw, a leading expert in the field of health and disease, is a Jeffrey Cheah professorial fellow in Gonville and Caius College, Cambridge. She is currently one of the principal UK scientists working on the European Prospective Investigation into Cancer and Nutrition, a Europe-wide project investigating the links between diet, lifestyle and cancer.

Appointed as a Commander of the order of the British Empire in 2003, Prof Khaw has been recognised for developing improved methods for collecting information on peoples diets and levels of exercise and relating this to the number of diagnosed cancer cases.

Prof Rema Hanna

A highly distinguished economist, Prof Hanna is the Jeffrey Cheah professor of South East Asia Studies and chair of the Harvard Kennedy School International Development Area, as well as the faculty director of evidence for policy design at Harvards Centre for International Development and the co-scientific director of the Abdul Latif Jameel Poverty Action Lab South East Asia office in Indonesia.

Her focus is on improving overall service delivery, understanding the impacts of corruption, bureaucratic absenteeism and discrimination against disadvantaged minority groups on delivery outcomes.

Prof Ketan J Patel

Prof Patel is a Jeffrey Cheah professorial fellow in Gonville and Caius College, Cambridge and the principal research scientist at the famous MRC Laboratory of Molecular Biology in the University of Cambridge.

His research, which focuses on the molecular basis of inherited genomic instability and the role it plays in the biology of stem cells, has been recognised through prestigious awards and prizes, including being elected as a fellow of the Royal Society of London, a member of the European Molecular Biology Organisation and a fellow of the Academy of Medical Sciences UK.

Prof John Todd

The Jeffrey Cheah fellow in medicine at Brasenose College, Oxford and professor of precision medicine, Prof Todd is a leading pioneer researcher in the fields of genetics, immunology and diabetes. His research areas include Type 1 diabetes genetics and disease mechanisms with the aim of clinical intervention.

In his former role as a professor of human genetics and a Wellcome Trust principal research fellow at Oxford, he helped pioneer genome-wide genetic studies, first in mice and then in humans.

Prof William Swadling

Prof Swadling, a Jeffrey Cheah professorial fellow, is a senior law fellow at Brasenose College, Oxford and Professor in the Law of Property in the Oxford University Law School.

An expert on the Law of Restitution, he is a contributor to Halsburys Laws of England, wrote the section on property in Burrows (ed) English Private Law and is widely cited in the British courts.

Prof William James

A Jeffrey Cheah professorial fellow emeritus and fellow in medicine at Brasenose College, Oxford, Prof James is a virologist with a background in genetics and microbiology.

As the professor of virology with the University of Oxford, he is the principal investigator at the Stem Cell Research Institute of Oxford, running a research lab studying HIV-macrophage biology using stem cell technology.

Prof Mark Wilson

Prof Wilson, the dean of Brasenose College, is a Jeffrey Cheah professorial fellow at the college and the professor of physical chemistry in the University of Oxfords physical and theoretical chemistry department.

The primary focus of his research interest is on the construction, development and application of relatively simple potential models to assess a wide range of systems with potentially unique properties.

Prof Jarlath Ronayne

Appointed in 2010 as the first Jeffrey Cheah distinguished professor, Prof Ronayne is a key member of Sunway Universitys board of directors and has played a pivotal role in establishing links between Sunway, Oxford and Cambridge.

Under his leadership, the Jeffrey Cheah Professorial Fellowships at Gonville and Caius College, Cambridge as well as Brasenose College, Oxford and the Jeffrey Cheah Scholar-in-Residence programmes in both colleges were established, alongside the prestigious Oxford University-Jeffrey Cheah Graduate Scholarship launched by the British High Commissioner in 2018. All these initiatives are in perpetuity.

Prof Sibrandes Poppema

A medical expert on Hodgkins disease, Prof Poppema has published more than 200 articles that have been cited more than 17,000 times.

The Jeffrey Cheah distinguished professor is also the co-owner of 12 patents and the founder of two biotechnology companies, as well as the advisor to the chancellor at Sunway University, especially on the establishment of a new medical school at the university.

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Setting the bar in education - The Star Online

Recommendation and review posted by Alexandra Lee Anderson

Site-specific glycan analysis of the SARS-CoV-2 spike – Science Magazine

SARS-CoV-2 spike protein, elaborated

Vaccine development for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on the trimeric spike protein that initiates infection. Each protomer in the trimeric spike has 22 glycosylation sites. How these sites are glycosylated may affect which cells the virus can infect and could shield some epitopes from antibody neutralization. Watanabe et al. expressed and purified recombinant glycosylated spike trimers, proteolysed them to yield glycopeptides containing a single glycan, and determined the composition of the glycan sites by mass spectrometry. The analysis provides a benchmark that can be used to measure antigen quality as vaccines and antibody tests are developed.

Science this issue p. 330

The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus 2019 (COVID-19) (1, 2), induces fever, severe respiratory illness, and pneumonia. SARS-CoV-2 uses an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry (3). The S protein is a trimeric class I fusion protein, composed of two functional subunits, responsible for receptor binding (S1 subunit) and membrane fusion (S2 subunit) (4, 5). The surface of the envelope spike is dominated by host-derived glycans, with each trimer displaying 66 N-linked glycosylation sites. The S protein is a key target in vaccine design efforts (6), and understanding the glycosylation of recombinant viral spikes can reveal fundamental features of viral biology and guide vaccine design strategies (7, 8).

Viral glycosylation has wide-ranging roles in viral pathobiology, including mediating protein folding and stability and shaping viral tropism (9). Glycosylation sites are under selective pressure as they facilitate immune evasion by shielding specific epitopes from antibody neutralization. However, we note the low mutation rate of SARS-CoV-2 and that as yet, there have been no observed mutations to N-linked glycosylation sites (10). Surfaces with an unusually high density of glycans can also enable immune recognition (9, 11, 12). The role of glycosylation in camouflaging immunogenic protein epitopes has been studied for other coronaviruses (10, 13, 14). Coronaviruses form virions by budding into the lumen of endoplasmic reticulumGolgi intermediate compartments (15, 16). However, observations of complex-type glycans on virally derived material suggests that the viral glycoproteins are subjected to Golgi-resident processing enzymes (13, 17).

High viral glycan density and local protein architecture can sterically impair the glycan maturation pathway. Impaired glycan maturation resulting in the presence of oligomannose-type glycans can be a sensitive reporter of native-like protein architecture (8), and site-specific glycan analysis can be used to compare different immunogens and monitor manufacturing processes (18). Additionally, glycosylation can influence the trafficking of recombinant immunogen to germinal centers (19).

To resolve the site-specific glycosylation of the SARS-CoV-2 S protein and visualize the distribution of glycoforms across the protein surface, we expressed and purified three biological replicates of recombinant soluble material in an identical manner to that which was used to obtain the high-resolution cryoelectron microscopy (cryo-EM) structure, albeit without a glycan-processing blockade using kifunensine (4). This variant of the S protein contains all 22 glycans on the SARS-CoV-2 S protein (Fig. 1A). Stabilization of the trimeric prefusion structure was achieved by using the 2P stabilizing mutations (20) at residues 986 and 987, a GSAS (Gly-Ser-Ala-Ser) substitution at the furin cleavage site (residues 682 to 685), and a C-terminal trimerization motif. This helps to maintain quaternary architecture during glycan processing. Before analysis, supernatant containing the recombinant SARS-CoV-2 S was purified by size exclusion chromatography to ensure that only native-like trimeric protein was analyzed (Fig. 1B and fig. S1). The trimeric conformation of the purified material was validated by using negative-stain EM (Fig. 1C).

(A) Schematic representation of the SARS-CoV-2 S glycoprotein. The positions of N-linked glycosylation sequons (N-X-S/T, where X P) are shown as branches (N, Asn; X, any residue; S, Ser; T, Thr; P, Pro). Protein domains are illustrated: N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM). (B) SDSpolyacrylamide gel electrophoresis analysis of the SARS-CoV-2 S protein (indicated by the arrowhead) expressed in human embryonic kidney (HEK) 293F cells. Lane 1: filtered supernatant from transfected cells; lane 2: flow-through from StrepTactin resin; lane 3: wash from StrepTactin resin; lane 4: elution from StrepTactin resin. (C) Negative-stain EM 2D class averages of the SARS-CoV-2 S protein. 2D class averages of the SARS-CoV-2 S protein are shown, confirming that the protein adopts the trimeric prefusion conformation matching the material used to determine the structure (4).

To determine the site-specific glycosylation of SARS-CoV-2 S, we used trypsin, chymotrypsin, and -lytic protease to generate three glycopeptide samples. These proteases were selected to generate glycopeptides that contain a single N-linked glycan sequon. The glycopeptides were analyzed by liquid chromatographymass spectrometry, and the glycan compositions were determined for all 22 N-linked glycan sites (Fig. 2). To convey the main processing features at each site, the abundances of each glycan are summed into oligomannose-type, hybrid-type, and categories of complex-type glycosylation based on branching and fucosylation. The detailed, expanded graphs showing the diverse range of glycan compositions are presented in table S1 and fig. S2.

The schematic illustrates the color code for the principal glycan types that can arise along the maturation pathway from oligomannose- to hybrid- to complex-type glycans. The graphs summarize quantitative mass spectrometric analysis of the glycan population present at individual N-linked glycosylation sites simplified into categories of glycans. The oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) is colored green, afucosylated and fucosylated hybrid-type glycans (hybrid and F hybrid) are dashed pink, and complex glycans are grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) and are colored pink. Unoccupancy of an N-linked glycan site is represented in gray. The pie charts summarize the quantification of these glycans. Glycan sites are colored according to oligomannose-type glycan content, with the glycan sites labeled in green (80 to 100%), orange (30 to 79%), and pink (0 to 29%). An extended version of the site-specific analysis showing the heterogeneity within each category can be found in table S1 and fig. S2. The bar graphs represent the mean quantities of three biological replicates, with error bars representing the standard error of the mean.

Two sites on SARS-CoV-2 S are principally oligomannose-type: N234 and N709. The predominant oligomannose-type glycan structure observed across the protein, with the exception of N234, is Man5GlcNAc2 (Man, mannose; GlcNAc, N-acetylglucosamine), which demonstrates that these sites are largely accessible to -1,2-mannosidases but are poor substrates for GlcNAcT-I, which is the gateway enzyme in the formation of hybrid- and complex-type glycans in the Golgi apparatus. The stage at which processing is impeded is a signature related to the density and presentation of glycans on the viral spike. For example, the more densely glycosylated spikes of HIV-1 Env and Lassa virus (LASV) GPC exhibit numerous sites dominated by Man9GlcNAc2 (2124).

A mixture of oligomannose- and complex-type glycans can be found at sites N61, N122, N603, N717, N801, and N1074 (Fig. 2). Of the 22 sites on the S protein, 8 contain substantial populations of oligomannose-type glycans, highlighting how the processing of the SARS-CoV-2 S glycans is divergent from host glycoproteins (25). The remaining 14 sites are dominated by processed, complex-type glycans.

Although unoccupied glycosylation sites were detected on SARS-CoV-2 S, when quantified they were revealed to form a very minor component of the total peptide pool (table S2). In HIV-1 immunogen research, the holes generated by unoccupied glycan sites have been shown to be immunogenic and potentially give rise to distracting epitopes (26). The high occupancy of N-linked glycan sequons of SARS-CoV-2 S indicates that recombinant immunogens will not require further optimization to enhance site occupancy.

Using the cryo-EM structure of the trimeric SARS-CoV-2 S protein [Protein Data Bank (PDB) ID 6VSB] (4), we mapped the glycosylation status of the coronavirus spike mimetic onto the experimentally determined three-dimensional (3D) structure (Fig. 3). This combined mass spectrometric and cryo-EM analysis reveals how the N-linked glycans occlude distinct regions across the surface of the SARS-CoV-2 spike.

Representative glycans are modeled onto the prefusion structure of the trimeric SARS-CoV-2 S glycoprotein (PDB ID 6VSB) (4), with one RBD in the up conformation and the other two RBDs in the down conformation. The glycans are colored according to oligomannose content as defined by the key. ACE2 receptor binding sites are highlighted in light blue. The S1 and S2 subunits are rendered with translucent surface representation, colored light and dark gray, respectively. The flexible loops on which the N74 and N149 glycan sites reside are represented as gray dashed lines, with glycan sites on the loops mapped at their approximate regions.

Shielding of the receptor binding sites on the SARS-CoV-2 spike by proximal glycosylation sites (N165, N234, N343) can be observed, especially when the receptor binding domain is in the down conformation. The shielding of receptor binding sites by glycans is a common feature of viral glycoproteins, as observed on SARS-CoV-1 S (10, 13), HIV-1 Env (27), influenza hemagglutinin (28, 29), and LASV GPC (24). Given the functional constraints of receptor binding sites and the resulting low mutation rates of these residues, there is likely selective pressure to use N-linked glycans to camouflage one of the most conserved and potentially vulnerable areas of their respective glycoproteins (30, 31).

We note the dispersion of oligomannose-type glycans across both the S1 and S2 subunits. This is in contrast to other viral glycoproteins; for example, the dense glycan clusters in several strains of HIV-1 Env induce oligomannose-type glycans that are recognized by antibodies (32, 33). In SARS-CoV-2 S, the oligomannose-type structures are likely protected by the protein component, as exemplified by the N234 glycan, which is partially sandwiched between the N-terminal and receptor binding domains (Fig. 3).

We characterized the N-linked glycans on extended flexible loop structures (N74 and N149) and at the membrane-proximal C terminus (N1158, N1173, N1194) that were not resolved in the cryo-EM maps (4). These were determined to be complex-type glycans, consistent with steric accessibility of these residues.

Whereas the oligomannose-type glycan content (28%) (table S2) is above that observed on typical host glycoproteins, it is lower than other viral glycoproteins. For example, one of the most densely glycosylated viral spike proteins is HIV-1 Env, which exhibits ~60% oligomannose-type glycans (21, 34). This suggests that the SARS-CoV-2 S protein is less densely glycosylated and that the glycans form less of a shield compared with other viral glycoproteins, including HIV-1 Env and LASV GPC, which may be beneficial for the elicitation of neutralizing antibodies.

Additionally, the processing of complex-type glycans is an important consideration in immunogen engineering, especially considering that epitopes of neutralizing antibodies against SARS-CoV-2 S can contain fucosylated glycans at N343 (35). Across the 22 N-linked glycosylation sites, 52% are fucosylated and 15% of the glycans contain at least one sialic acid residue (table S2 and fig. S3). Our analysis reveals that N343 is highly fucosylated with 98% of detected glycans bearing fucose residues. Glycan modifications can be heavily influenced by the cellular expression system used. We have previously demonstrated for HIV-1 Env glycosylation that the processing of complex-type glycans is driven by the producer cell but that the levels of oligomannose-type glycans were largely independent of the expression system and are much more closely related to the protein structure and glycan density (36).

Highly dense glycan shields, such as those observed on LASV GPC and HIV-1 Env, feature so-called mannose clusters (22, 24) on the protein surface (Fig. 4). Whereas small mannose-type clusters have been characterized on the S1 subunit of Middle East respiratory syndrome (MERS)CoV S (10), no such phenomenon has been observed for the SARS-CoV-1 or SARS-CoV-2 S proteins. The site-specific glycosylation analysis reported here suggests that the glycan shield of SARS-CoV-2 S is consistent with other coronaviruses and similarly exhibits numerous vulnerabilities throughout the glycan shield (10). Last, we detected trace levels of O-linked glycosylation at Thr323/Ser325 (T323/S325), with over 99% of these sites unmodified (fig. S4), suggesting that O-linked glycosylation of this region is minimal when the structure is native-like.

From left to right, MERS-CoV S (10), SARS-CoV-1 S (10), SARS-CoV-2 S, LASV GPC (24), and HIV-1 Env (8, 21). Site-specific N-linked glycan oligomannose quantifications are colored according to the key. All glycoproteins were expressed as soluble trimers in HEK 293F cells apart from LASV GPC, which was derived from virus-like particles from Madin-Darby canine kidney II cells.

Our glycosylation analysis of SARS-CoV-2 offers a detailed benchmark of site-specific glycan signatures characteristic of a natively folded trimeric spike. As an increasing number of glycoprotein-based vaccine candidates are being developed, their detailed glycan analysis offers a route for comparing immunogen integrity and will also be important to monitor as manufacturing processes are scaled for clinical use. Glycan profiling will therefore also be an important measure of antigen quality in the manufacture of serological testing kits. Last, with the advent of nucleotide-based vaccines, it will be important to understand how those delivery mechanisms affect immunogen processing and presentation.

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Site-specific glycan analysis of the SARS-CoV-2 spike - Science Magazine

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Demand for Syndromes of Progressive Ataxia and Weakness Disorders Market to Witness Rapid Surge – Lake Shore Gazette

Ataxia is a neurological condition, characterized by lack of voluntary coordination of muscle movement. Ataxia causes head trauma, stroke, Transient Ischemic Attack (TIA), tumor and toxic reaction. Progressive ataxia and weakness disorders are related to damage, degeneration or loss of neurons of the brain which leads to muscle coordination disability.

The global market for treatments of syndromes of progressive ataxia and weakness disorders is categorized based on various drugs used for treatment of progressive ataxia syndromes, drugs for progressive weakness syndromes and by technology. The progressive ataxia syndrome segment is further sub-segmented into major diseases, such as Friedreichs ataxia, Gertsman-Straussler-Scheinker disease and Machado-Joseph disease. The progressive weakness syndrome segment includes amyotrophic lateral sclerosis, hereditary spastic paraplegia, hereditary neuropathies, progressive bulbar palsy and multiple sclerosis. The technology segment is further sub-segmented into small molecules based therapies and monoclonal antibody.

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Globally, treatments for syndromes of progressive ataxia and weakness disorders market are growing due to novel drug development and rapid technological advancement for treatment of progressive ataxia and weakness disorders. Some of the major technological advancement involved in growth of the market are protein mis-folding, gene mutation and stem cell therapy. In addition, increased collaborations between industry players for development of new therapies is a key trend for the market.

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Demand for Syndromes of Progressive Ataxia and Weakness Disorders Market to Witness Rapid Surge - Lake Shore Gazette

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Beyond the cell factory: Homeostatic regulation of and by the UPRER – Science Advances

Abstract

The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is responsible for a large amount of protein and lipid synthesis. As a membrane-bound organelle, the ER has a distinct environment that is ideal for its functions in synthesizing these primary cellular components. Many different quality control machineries exist to maintain ER stability under the stresses associated with synthesizing, folding, and modifying complex proteins and lipids. The best understood of these mechanisms is the unfolded protein response of the ER (UPRER), in which transmembrane proteins serve as sensors, which trigger a coordinated transcriptional response of genes dedicated for mitigating the stress. As the name suggests, the UPRER is most well described as a functional response to protein misfolding stress. Here, we focus on recent findings and emerging themes in additional roles of the UPRER outside of protein homeostasis, including lipid homeostasis, autophagy, apoptosis, and immunity.

Multicellular organisms face a constant barrage of stresses that warrant an effective response, coordinated across diverse tissues. Each cell or tissue must thus be capable of perceiving stresses and signaling distal cells to respond accordingly to mitigate perturbations in cellular function and homeostasis. Furthermore, the distinct membrane-bound environments of the cell require these stress responses to be compartment specific. To maintain homeostasis of these microenvironments, cells have evolved several subcellular stress responses, including the cytoplasmic heat shock response (HSR), the endoplasmic reticulum (ER) unfolded protein response (UPRER), and the mitochondrial unfolded protein response (UPRmt) (13). Of these responses, the ERs central function in biosynthesis, folding, and modification of membrane-bound and secreted proteins and its major role in lipid synthesis place particular interest on the UPRER. This interest is highlighted by the fact that defects in ER function are significantly associated with obesity, diabetes, cancer, and age-onset neurodegenerative disease (4, 5).

There are three primary branches of the UPRER, which enable the ER to maintain normal levels of protein folding, protein secretion, and lipid homeostasis. Each arm of the UPRER consists of a transmembrane protein containing a luminal-facing domain and transmembrane helix, which act as sensors for induction of a nuclear signal upon detection of ER stress (Fig. 1). The best characterized of the three UPRER branches involves an endonuclease, inositol-requiring protein 1 (IRE1 in mammals, IRE-1 in Caenorhabditis elegans, and Ire1p in Saccharomyces cerevisiae. Note: All gene and protein names will use nomenclature pertinent to the organism, and human nomenclature is used as a general terminology when no organism is specified), and a transcription factor, X-box binding protein 1 (XBP1 in mammals, XBP-1 in C. elegans, and Hac1p in S. cerevisiae). In this branch, unfolded protein stress or lipid disequilibrium is sensed from the ER-localized IRE1, which then undergoes homodimerization and autophosphorylation. This activates IRE1s cytosolic endonuclease domain to splice a specific intron from the mRNA of XBP1u to create XBP1s. The spliced mRNA is translated into XBP1s, which translocates into the nucleus to mediate expression of protein degradation, protein folding, and lipid metabolism gene targets (2, 6). IRE1 also plays an important role in regulating mRNA levels through regulated IRE1-dependent decay (RIDD). A majority of the identified RIDD mRNA targets encode proteins with signal peptides and transmembrane domains, including several secreted components of the insulin secretory pathway in cells and mucin 2 in secretory goblet cells, whose reduced translation is expected to reduce the protein-folding load on the ER under conditions of ER stress or damage (79).

There are three branches of UPRER, each consisting of a transmembrane protein with a luminal-facing sensor for damage, which then signals to the nucleus through a unique transcription factor. When IRE1 senses misfolded protein or lipid stress in the ER, it homodimerizes, is autophosphorylated, and promotes splicing of XBP1u mRNA to XBP1s which is translated into functional XBP1s, acting as a transcription factor to turn on genes important for restoring ER homeostasis. Similarly, PERK and ATF6 are activated under ER stress. When PERK is activated, it also oligomerizes, causing phosphorylation of eIF2 to inhibit global translation. There is also downstream activation of ATF4, which promotes the expression of ER-restoring genes that escape down-regulation via eIF2. Unlike the other two ER stress sensors, ATF6 is proteolytically cleaved under ER stress, which causes translocation to the Golgi for further processing, allowing ATF6 to function as a transcription factor.

The other branches of the UPRER have different mechanisms of action, namely, the (i) global reduction of protein translation via eIF2 downstream of protein kinase RNA-like ER kinase (PERK in mammals and PEK-1 in C. elegans) and (ii) the proteolytic cleavage of an ER-resident protein, which translocates to the Golgi under stress to become a proteostasis-promoting transcription factor, activating transcription factor 6 (ATF6 in mammals and ATF-6 in C. elegans) (2, 6). Similar to IRE1, PERK undergoes homodimerization and phosphorylation in response to unfolded proteins and lipid disequilibrium in the lumen. This leads to phosphorylation of eIF2, which induces a global down-regulation of translation. However, critical mRNA species escape this translational down-regulation, including the activation of transcription factor 4 (ATF4 in mammals and ATF-4 in C. elegans), which is up-regulated during ER stress to promote the integrated stress response through remodeling of metabolic and translational programs (10). In addition, ATF4 can promote apoptosis during sustained ER stress by up-regulating CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP).

The third arm of the UPR is initiated by ATF6, a type II ER transmembrane protein that translocates to the Golgi upon activation. During stress, the luminal domain of ATF6 loses its association with BiP/GRP78 (HSP-4 in C. elegans), which causes translocation of ATF6 into the Golgi. Once in the Golgi, Golgi-resident site 1 protease (S1P) and site 2 protease (S2P) cleave ATF6, allowing the N-terminal cytosolic fragment to translocate into the nucleus and act as a transcription factor to up-regulate target genes, including protein disulfide isomerase (PDI), XBP1, and CHOP (1113).

Dysregulation of the UPRER is a common feature of many diseases, including neurodegeneration, metabolic disease, and cancer. During the aging process, UPRER activation also becomes dysregulated across multiple organisms. For example, in C. elegans, the capacity to activate XBP-1mediated UPRER in response to protein misfolding stress declines sharply during the aging process (14). Similarly, in aged mice, expression of genes involved in ER quality control show marked decline in the brain (15, 16). The decreased function of the UPRER during aging can lead to the accumulation of damaged and aggregated proteins, which contribute to proteotoxicity and eventual cell death (17). Conversely, up-regulation of ER chaperones can protect cells during stress (18, 19), and hyperactivation of the UPRER can have direct impacts on life span and healthspan: Overexpression of xbp-1s in C. elegans extends life span and stress resistance (14), and increased PERK-eIF2 signaling protects neurons from stress associated with misfolded proteins (20, 21). Many of these studies focus primarily on chaperones and other mechanisms involved in restoring protein homeostasis. However, it is clear that there are other critical downstream targets of the transcription factors involved in up-regulating UPRER. This review touches on these core machineries outside of protein homeostasis and highlights the open-ended questions involved in how stress affects other functions of the ER, such as lipid and redox homeostasis.

Beyond the UPRER, there are several other mechanisms involved in maintaining ER homeostasis. Given the major role of the ER in protein synthesis, there are limited proteases that function within the ER. Therefore, proteins that are beyond repair, such as terminally misfolded proteins, are first extracted from the ER by adenosine triphosphatedriven motors and targeted for proteasomal degradation through ER-associated degradation (ERAD). In yeast, where most of the ERAD components have been originally described, transmembrane protein complex including the ubiquitin ligases Hrd1p and Doa10p recognize misfolded proteins and tag them for degradation (22, 23). Upon poly-ubiquitylation via the ERAD machinery, the AAA+ adenosine triphosphatase (ATPase) Cdc48p (p97 or valosin-containing protein in humans) drives extraction of the proteins from the ER into the cytosol, where it is subsequently degraded by the proteasome (24). ERAD also plays an important role in maintaining protein quantity control by tagging excess or unnecessary proteins for degradation through similar mechanisms (25, 26). When accumulation of damaged proteins in the ER has exceeded the repair capacity of ERAD, portions of the organelle can be specifically targeted for large-scale degradation through autophagy (ER-phagy). ER-phagy is capable of clearing ERAD-resistant proteins or other ER components, such as lipids, which cannot be cleared by conventional quality control machineries but are generally subject to autophagy through Vps34p/beclin-1dependent machinery (27). It would be of great interest to understand whether ERAD and ER-phagy are critical for maintaining ER function outside of its proteome. It is possible to imagine that eliminating damaged ER via autophagy will also remove toxic lipid species, but can ERAD impose a similar benefit to lipids and other nonprotein components of the ER?

Here, we focus primarily on the UPRER with specific emphasis on noncanonical roles of UPRER outside of protein quality control. For a more thorough review on ER quality control machineries outside of UPRER, refer to (1, 28, 29).

Lipids are synthesized and metabolized within multiple organelles; however, specific functions are compartmentalized within organelles to maintain lipid homeostasis. For example, initial fatty acid synthesis primarily occurs in the mitochondria and cytoplasm. Subsequent fatty acid elongation then occurs within the mitochondria, cytoplasm, and ER (30, 31). More complex lipids such as ether lipids are produced by the peroxisome, while sterols, phospholipids, and neutral lipids are synthesized by the ER. Thus, many critical enzymes for lipid metabolism reside in the ER, making the ER a critical hub for lipid homeostasis and a primary source of membrane lipids for all other organelles (32, 33).

Since the ER serves as a critical organelle in regulation of lipid homeostasis, key sensors monitor lipid quality within the ER. These sensors are the same UPRER transmembrane proteins involved in protein homeostasis: IRE1, PERK, and ATF6. Adjacent to their transmembrane helices, IRE1 and PERK contain an amphipathic helix capable of sensing general ER membrane imbalances and can activate the UPRER independent of their luminal unfolded protein-sensing domains (34, 35). Within the transmembrane domain of ATF6, a sphingolipid-sensing motif is able to trigger ATF6 activation upon accumulation of dihydrosphingosine or dihydroceramide, also independent of proteotoxic stress (36). In combination with basal lipid metabolism transcription factors, these proteins play an integral role in maintaining lipid homeostasis. Activation of UPRER alters the expression of many lipid metabolism genes. For example, PERK/eIF2 phosphorylation activates sterol regulatory elementbinding protein-1c (SREBP-1c) and SREBP-2, master transcription factors that regulate enzymes of lipogenic pathways (37). Mice with compromised eIF2 signaling down-regulate lipogenesis and displayed reduced high-fat diet (HFD)induced fatty livers (38). Furthermore, XBP1s directly up-regulates lipogenic genes, including Dgat2, Scf1, and Acc2, while deletion of Xbp1 results in hypocholesterolemia and hypotriglyceridemia of the liver (39). Last, large-scale sequencing studies in C. elegans found that a large subset of genes induced by IRE-1, XBP-1, PEK-1, and ATF-6 under conditions of ER stress were involved in lipid and phospholipid metabolism (40).

Two recent, complementary studies found that constitutive activation of UPRER downstream of xbp-1s resulted in notable lipid depletion in C. elegans. The original study from our laboratory describing xbp-1s overexpression in C. elegans identified that overexpression of xbp-1s in neurons was sufficient to elicit nonautonomous UPRER activation in peripheral tissue to promote whole-organism life-span extension (14). However, overexpression in other tissues either failed to elicit the same response or was detrimental in some other cases, suggesting that neurons were specialized in sending a specific and beneficial stress signal to other cells. Another unexpected study from our laboratory found that glia could signal a similar beneficial signal to the periphery (41).

Following this work, neuron-specific overexpression of xbp-1s was found to result in whole-animal depletion of lipids via two mechanisms: (i) up-regulation of lysosomal lipases and desaturases, which resulted in decreased triglycerides and increased oleic acid levels (42), and (ii) activation of lipophagy via a conserved RME-1/RAB-10/EHBP-1 (receptor mediated endocytosis-1/ras- related GTP binding protein-10/EH domain binding protein-1) complex, which depletes neutral lipids and decreases lipid droplet size and number, a phenomenon described by our work (Fig. 2, left) (43). When xbp-1s is overexpressed in neurons, both protein homeostasis and lipid metabolism are activated in peripheral tissue (14, 43). Perturbations of either protein homeostasis or lipid metabolism suppress the beneficial effects of neuronal xbp-1s overexpression on life span and ER stress resistance, suggesting that both are essential components downstream of xbp-1s to promote ER quality control and organismal health. However, the most notable finding in the latter study is that the beneficial effects of lipid depletion on animal physiology can be uncoupled from protein homeostasis. Overexpression of ehbp-1 is sufficient to drive lipid depletion and life-span extension but does not promote chaperone induction, suggesting that these two mechanisms can be uncoupled. In the former study, changes in lipid profiles caused by xbp-1s overexpression in neurons were sufficient to drive improvements in protein homeostasis. Specifically, supplementation with oleic acid decreased toxicity associated with ectopic polyQ40 expression, suggesting that changes in lipid homeostasis are sufficient to improve protein quality control even in the absence of chaperone induction. Since the ER is composed of both integral lipids and proteins, it is likely that promoting overall ER quality drives global organelle homeostasis, although further studies are required to understand the cross communication of lipid and protein quality control machineries within the ER. Whether this is indirect (i.e., the decreased burden of maintaining lipid homeostasis allows the ER to divert all its energy to protein quality control machineries) or direct (i.e., ER lipid health can directly alter protein folding via a still unknown molecular pathway) is still under investigation. In addition, the specific signal originating from neurons to drive these seemingly separable changes in the periphery also remains to be discovered.

In C. elegans (left), overexpression of xbp-1s in neurons promotes two distinct changes to ER homeostasis in peripheral tissue (intestine): increased protein homeostasis by up-regulation of chaperones and increased lipid metabolism through mobilization of lipids via lipases, desaturases, and increased lipophagy. Both the increase in protein folding and decreased lipids are essential for the life-span extension found in this paradigm. Ectopic expression of xbp-1s in glia has also been shown to promote peripheral protein homeostasis and extend life span, although a role in glial signaling in lipid homeostasis has yet to be described. A similar phenomenon was also found in mice (right), where overexpression of Xbp1s in Pomc neurons (or simply activating Pomc neurons via olfactory exposure to food) is sufficient to drive UPRER in peripheral tissue. Specifically, XBP1s in POMC neurons promotes XBP1s and mTOR signaling in hepatocytes and adipose tissue, resulting in increased metabolic health, including resistance to diabetes and obesity. As UPRER has been shown to be critical in proper muscle and B cell function, it would be of great interest to investigate whether neuronal XBP1s can signal to elicit a beneficial effect in these and other cell types.

A similar communication from neurons to peripheral tissue is observed in vertebrates. When Xbp1s is overexpressed in Pomc neurons of the hypothalamus of mice, the UPRER is up-regulated and has beneficial impacts on metabolic physiology (e.g., improved glucose levels, improved insulin sensitivity, and protection against HFD-induced obesity) (Fig. 2, right) (44). In this model, Xbp1s increases Pomc neuronal activity, which in turn increases energy expenditure by promoting brown adipose tissue thermogenesis and browning of white adipose tissue, which results in an overall decrease in fat mass and body weight, consistent with the findings in C. elegans. Conversely, mice with Xbp1 deleted only in neurons or glia are more susceptible to diet-induced obesity and exhibit elevated levels of insulin and leptin in response to HFD (45). In mice, food perception (i.e., smelling of food) was sufficient to drive a Pomc neuron response to activate hepatic mammalian target of rapamycin (mTOR) and XBP1 signaling to promote metabolic homeostasis (46). Mice with olfactory exposure to food were able to phenocopy Xbp1s overexpression in Pomc neurons, driving peripheral Xbp1 activation and its downstream beneficial effects on animal physiology. Both protein homeostasis and lipid homeostasis are activated via peripheral Xbp1 activation (e.g., hepatic tissue activation upon receiving cues from Pomc neurons), and it is unclear whether these two mechanistic pathways can be uncoupled in mammalian models as was found in C. elegans.

Determining whether promoting chaperones and overall protein handling in the ER can alter lipid homeostasis and vice versa would be of great interest to understanding the independent roles that lipids and proteins have on mammalian organismal health. Is enhancing lipophagy through EHBP1 sufficient to drive ER stress resistance and organismal healthspan and life span in mammals similar to C. elegans? Do there exist divergent nodes of protein and lipid homeostasis downstream of XBP1s, or are these downstream mechanisms overlapping in higher eukaryotes? Under disease conditions, is loss of a single node of XBP1s signaling sufficient to drive pathogenesis? These questions are critical to develop novel therapeutic intervention for diseases that cause dysregulation of UPRER.

While the activation of the UPRER has many implications in organismal health and life span, persistent activation of the UPRER is associated with several metabolic diseases. Chronic UPRER activation is often observed in the liver or adipose tissue of models of obesity, nonalcoholic fatty liver disease, and diabetes (47). Moreover, ER stress within the brains metabolic control center, the hypothalamus, has been shown to contribute to metabolic changes that promote weight gain and insulin resistance in mice, hallmark symptoms of obesity (6, 48). A major feature of obesity is increased free fatty acids in circulation, which have been linked to UPRER activation in several models (49, 50). Excessive accumulation of lipids can cause metabolic abnormalities and initiate cell death in response to lipotoxicity, often linked to chronic ER stress and defects in UPRER signaling. Specifically, saturated fatty acids, such as palmitate, activate the UPRER and cause detrimental effects in pancreatic , liver, adipose, and muscle cells.

In primary rat cells, exposure to palmitate results in increased phosphorylation of eIF2 through PERK activation, increased Xbp1s splicing, and increased ATF4 activity (5153). Elevated levels of palmitate can result in excessive palmitoylation of proteins, which induce ER stress and activate caspase activity, causing cell death. In addition, excess palmitate can also cause lipotoxicity and ER dysfunction by altering the composition and membrane fluidity of the ER by changing phospholipid composition (54), promoting ceramide accumulation (55), and altering sphingolipid metabolism (56). Regardless of the mechanism, the chronic activation of the ER stress response promotes cell death through the induction of apoptosis, which often includes the hyperactivation of cytokines, including interleukin-1 (IL-1), interferon-, tumor necrosis factor (TNF), and nuclear factor B (NF-B) [reviewed in (57)].

Similarly, ER stress through exposure to saturated fatty acids is a major contributing factor in liver lipotoxicity. In several liver cell lines, including HepG2 hepatoma and L02 immortalized liver cells, exposure to saturated fatty acids resulted in activation of PERK and up-regulation of its downstream targets such as ATF4 and CHOP (58). Suppression of PERK activation or reducing ER stress load via overexpression of BiP was sufficient to reduce palmitate-induced death (58, 59). Liver cell exposure to palmitic acid results in aberrant phospholipid metabolism and increased membrane saturation (60). Alterations in the ER lipid composition and fluidity inhibit ER Ca++ signaling (61), which can result in aberrant mitochondrial metabolism and increased reactive oxygen species (ROS) production, causing further cellular toxicity (62). Restoring ER lipid composition through conversion of saturated lipid species into unsaturated fatty acylcoenzyme As (CoAs) by overexpressing catalytic enzymes, such as Lpcat3, or restoring Ca++ homeostasis by overexpression of sarco-ER calcium ATPase reduces lipotoxicity in liver cells and can improve hepatic function in obese individuals (61, 63). Last, lipid overload impairs autophagic flux in murine models and human patients with nonalcoholic fatty liver disease, suggesting a functional role for autophagy in preventing ER stressmediated apoptosis (64).

Although less understood, muscle cells are also sensitive to lipid-induced ER stress. Mice fed an HFD showed up-regulation of Xbp1 splicing, BiP, and ATF4/CHOP in skeletal muscle (65), while myotubes exposed to high levels of palmitate induced ATF4 and XBP1 activity (66). Prolonged lipotoxicity in muscle cells results in increased inflammation and ER stress, which can promote insulin resistance. Overexpression of stearoyl-CoA desaturase 1 (SCD1), a key regulator in lipid metabolism, can restore lipid homeostasis and reduce inflammatory cytokine expression, ultimately preventing insulin resistance in myotubes (66). However, a separate study in human and mouse cells showed that restoring ER homeostasis in palmitate-treated muscle cells did not restore insulin signaling, suggesting that palmitate-induced ER stress may not be the cause of reduced insulin signaling (67). Another study in human patients on a high-fat, hypercaloric diet showed similar contradicting results. While patients on HFD exhibited glucose intolerance, skeletal muscle biopsies failed to show an increase in ER stress markers, including XBP1, BiP, or PERK (68). Thus, further research is necessary to elucidate the connection between lipotoxicity and ER homeostasis in skeletal muscle cells.

Despite these controversies, a recent study in mice showed an interesting role for skeletal muscle in signaling lipotoxicity to other cells. Here, muscle-specific knockout of the lipid dropletassociated protein, perilipin 5, caused an increase in fatty acid oxidation and reduced ER stress in muscle cells. This resulted in whole-body glucose intolerance and insulin resistance due to reduced secretion of fibroblast growth factor 21 from both skeletal and liver cells, highlighting a critical cross-talk between muscle and liver in ER lipid homeostasis (69).

Overall, it is clear that the UPRER plays a critical role in regulation of lipid homeostasis and metabolic state of the organism. Still to be investigated is whether the impact of UPRER activity serves to be beneficial or detrimental to organismal health. While many studies have highlighted a beneficial effect of UPRER activation in neurons (14, 41, 42, 44), whole-organism xbp-1s overexpression has no beneficial effect on life span in C. elegans (14). Thus, it is possible that increased UPRER signaling can be detrimental in some tissue. Next, we describe the potential detrimental impacts of a sustained UPRER.

Despite many studies providing evidence for UPRER providing a beneficial role in clearing damage, sustained and unresolved ER stress can result in activation of apoptosis. Hence, chronic and irreversible UPRER induction can contribute to pathophysiological processes involved in a number of diseases, including neurodegeneration. In unresolved ER stress, the PERK-ATF4 axis of the UPRER induces the transcriptional activation of proapoptotic machinery, including C/EBP-homologous protein CHOP. CHOP then promotes the down-regulation of the antiapoptotic factor, B cell lymphoma 2 (BCL2), and activation of proapoptotic genes, thus inducing the core mitochondrial apoptosis machinery through BCL2-associated X protein (BAX) and BCL2-antagonist/killer 1 (BAK) (70).

Under certain conditions, chronic ER stress can also regulate cell death decisions by influencing several mitogen-activated protein kinase (MAPK)signaling components, including extracellular signalregulated kinase (ERK), p38 MAPK, and JUN N-terminal kinase (JNK) (Fig. 3) (71, 72). For example, ER stressinduced JNK activation is thought to initiate a proapoptotic pathway. Under ER stress, IRE oligomerizes, activating its kinase domain and increases interaction with TNF receptorassociated factor 2 (TRAF2), which activates JNK via induction of apoptosis signalregulating kinase 1 (ASK1). IRE1-TRAF2 promotes ASK1 oligomerization and autophosphorylation, which is required for its kinase activity to promote JNK signaling (73). Activation of JNK signaling can promote cell death by promoting de novo synthesis of death receptors and their ligands and by targeting components of the BCL2 family to initiate apoptosis (74). Inhibiting the downstream activation of JNK has been shown to promote resistance to ER stressinduced cell death: In human pancreatic cells, inhibition of JNK significantly decreased eIF2 activity and promoted cell viability under ER stress (75); Ask1/ mice showed reduction in JNK activation and decreased apoptosis under ER stress (76), and phosphorylation of ASK1 on Ser83 decreased its activity, promoting prosurvival by reducing apoptosis (77). In addition to the IRE1-TRAF2-ASK1 pathway, JNK can also be activated by the PERK axis of UPRER through CHOP. CHOP expression promotes the release of Ca++ from the ER, which also activates ASK1 through Ca++/calmodulin-dependent protein kinase II (CaMKII) (78). JNK activation through CaMKII-ASK1 promotes apoptosis through increased cell surface localization of the death receptor Fas, and in vivo knockout of CaMKII can suppress apoptosis induced via ER stress (79).

Functionally, the UPRER serves as a quality control mechanism to restore ER form and function under conditions of stress. However, under sustained and unresolved ER stress, UPRER can actually promote cell death through apoptosis. For example, sustained PERK signaling can promote the activation of CHOP through ATF4, which activates proapoptotic signals. The other branches of UPRER can also modulate MAPK signaling, which feeds into cell survival or apoptotic cues in various ways. For example, IRE-1 can activate both prosurvival signals through activation of ERK1/2 and proapoptotic signals through JNK depending on the ER stress conditions. Beyond the UPRER, extracellular cues can promote cell survival under ER stress. Specifically, the cell surface hyaluronidase, TMEM2, cleaves highmolecular weight hyaluronic acid (HMW HA) into lowmolecular weight hyaluronic acid (LMW HA), which acts as a ligand to the CD44 receptor and activates downstream p38 and ERK1/2 prosurvival signals.

In contrast to JNK signaling, activation of ERK1/2 signaling serves as a prosurvival cue under ER stress. As a primary signaling molecule downstream of almost all growth factors, ERK1/2 promotes cell survival under numerous stress stimuli by promoting transcriptional activation of several prosurvival proteins, including BCL2 (80). Moreover, ERK1/2 activation under ER stress is dependent on IRE1. In gastric cancer cells, IRE1 knockdown decreased ERK1/2 signaling under ER stress, which results in decreased BiP levels and subsequent induction of cell death (81). In mouse embryonic fibroblasts, IRE1 also regulates ERK1/2 signaling by regulating the pool of the Src homology 2/Src homology 3 domaincontaining adaptor Nck. Under basal conditions, ER-associated Nck suppresses ERK1 signaling, but upon exposure to ER stress, Nck dissociates from the ER membrane, eliciting IRE1-dependent ERK1 activation to promote cell survival (82). However, how IRE1 promotes the activation of ERK1 is still unclear.

ERK1/2 hyperactivation is also found in numerous cancers and is a target for therapeutic intervention (83). Several human melanoma cell lines have been shown to be protected from therapeutic interventions that promote ER stressinduced apoptosis due to increased ERK1/2 signaling in these cancers. In some cases, inhibition of ERK1/2 signaling increased sensitivity of cancer cells to ER stressinduced cell death, introducing combined ERK1/2 inhibition and ER stress as a potential therapeutic intervention for these cancers, including melanoma (84).

MAPK signaling does not only function downstream of UPR activation but can also promote UPRER signaling. For example, p38 MAPK can phosphorylate two serine residues found in CHOP, increasing the activity of its transactivation domain (85). While the phosphorylation of these serine residues by p38 was not critical for the DNA binding activity of CHOP, they had notable implications in its association with binding partners required to promote cell death machinery (86). In cardiomyocytes, ATF6 has also been shown to be a direct substrate for phosphorylation by p38 (87). Sustained p38 activity increased ATF6 phosphorylation and promotes its downstream signaling, including the induction of BiP (88, 89).

A recent study from our laboratory elucidated a role for MAPK signaling in maintaining ER stress resistance independent of the UPRER (90). Through whole-genome CRISPR-Cas9 screening in karyotypically normal fibroblasts, the cell surface hyaluronidase transmembrane protein 2 (TMEM2) was identified as a novel regulator of ER homeostasis. Specifically, overexpression of TMEM2 increased resistance to ER stress through ERK and p38 MAPK signaling. While the exact signaling cascade is unknown, it is proposed that the lowmolecular weight product of hyaluronic acid produced by TMEM2 converges on the CD44 receptor to activate ERK and p38-dependent cell survival under ER stress. Intriguingly, overexpression of human TMEM2 in C. elegans was sufficient to extend life span by more than 20% by preventing the age-associated decline in innate immunity (immunosenescence), similarly dependent on ERK/p38 (PMK-1/MPK-1 in C. elegans). Most of the cells in the adult nematode are postmitotic, and MAPK signaling does not play a role in regulating apoptosis in the adult. Rather, the central role of MAPK signaling is in regulating innate immunity (91). Perhaps, most notable in the study was that the beneficial effects of TMEM2 were completely independent of all three branches of UPRER. Therefore, despite numerous studies highlighting notable overlap between UPRER and MAPK signaling modalities, it is clear that there exist mutually exclusive mechanisms of modulating cell survival under conditions of ER stress.

Beyond apoptosis, chronic activation of PERK signaling can result in sustained repression of translation through eIF2, which can also be detrimental. For example, in animal models, hyperactivation of PERK promotes synaptic failure and neuronal death in prion disease mouse models, which suggests that decreasing UPRER activity could be a potential therapeutic intervention by restoring protein synthesis in neurons (58). In triple-negative breast cancers, hyperactivation of XBP1 can also promote tumor growth, and inhibition of IRE1/XBP1 was shown to be beneficial (59). Thus, it is clear that UPRER signaling is complex and context specific, highlighting the importance of dissecting the molecular mechanisms downstream of UPRER activation for therapeutic intervention.

ER stress is commonly found in inflammatory diseases, such as diabetes, atherosclerosis, and inflammatory bowel disease (92). Accumulating evidence links the activation of the UPRER in inflammatory signaling cascades, including the activation of cytokine release (93). In addition, several studies indicate that inflammation itself augments ER stress responses (Fig. 4). For example, exposure to proinflammatory cytokines, such as TNF, IL-1, and IL-6, induced ER stress, promoted XBP1s expression, and activated UPR in mouse livers and fibrosarcoma cells (94, 95). In addition, lipopolysaccharide (LPS) stimulation resulted in the activation of XBP1s, ATF4, and CHOP in mice (96). These studies strongly link the connection between ER stress and immunity.

The immune response and the UPRER have both been shown to affect the other. Mounting an immune response requires the synthesis of many proteins, including several secreted factors, which makes a functional ER imperative during pathogenic infection. Thus, under exposure to pathogens, UPRER is activated to promote protein homeostasis. In addition, to avoid cell death, immune signals may dampen the PERK arm to inhibit apoptosis. UPRER components can also alter immunity through IRE1-mediated activation of TRAF2, which can promote cytokine signaling through NF-B or directly alter transcription of immune response genes through p38 MAPK signaling.

Perhaps the first identified role of UPRER in the immune system was in the development of specific immune cells. For example, XBP1 is critical for the development of immunoglobulin-secreting plasma cells, such that mice lacking Xbp1 fail to mount antibody responses, have decreased levels of all immunoglobulins, and are more susceptible to infections that are normally cleared by antibody-mediated immune responses (97). Subsequent studies have shown that functional B cells splice Xbp1 mRNA and up-regulate UPR target genes, including BiP, upon exposure to LPS (98, 99). It is likely that the massive induction of UPR in B cells is critical to expand the ER and promote protein synthesis to meet the new secretory demands of a mature B cell (100). Both XBP1 activity and ATF6 activity reach maximal levels once Ig synthesis and secretion are induced in B lymphocytes (101). PERK is not activated upon LPS stimulation, and B cells lacking Perk develop normally and are fully capable of Ig synthesis and antibody secretion, providing further evidence that the purpose of UPRER activation in B cells is primarily to meet the increased secretory demands of these cells (102).

Similar to B cells, T cell differentiation is also highly dependent on a functional UPR. During viral or bacterial infection, expansion of antigen-presenting CD8+ T cells requires splicing of Xbp1 mRNA downstream of IL-2 signals. Unlike B cells, T cells exhibit increased Atf4 mRNA, suggesting that the PERK/eIF2 pathway is also activated during T cell differentiation (103). Xbp1 splicing is also critical in maintaining dendritic cells (professional antigen-presenting cells), as loss of XBP1 leads to reduced numbers due to increased apoptosis of dendritic cells, whereas overexpression of Xbp1s promotes their survival (104). In addition to promoting survival in these cell types, ER stress also plays a critical role in antigen presentation, although the exact mechanism is not yet understood (105, 106). Increased levels of triglycerides have been found in dendritic cells in both mice and human patients with tumors (107, 108). Lipid accumulation occurs in dendritic cells due to up-regulation of receptors involved in extracellular lipid uptake, which has detrimental effects in dendritic cell function (109). It would be of particular interest to determine whether hyperactivation of XBP1 can promote lipid depletion in dendritic cells similar to the neuronal XBP1 signaling paradigms described in mice and nematodes. Can Xbp1 overexpression promote dendritic cell survival and function by preventing accumulation of triglycerides? Pharmacological normalization of lipid levels on dendritic cells restored their functional activity and promoted immune response (109).

UPRER also affects innate immunity. Exposure to ER stress activates many inflammatory signaling cascades, including NF-B, which is considered a major mechanism for inducing the innate immune response. Under ER stress, IRE1 interacts with inhibitor of nuclear factor B (IB) kinase through TRAF2, which enhances TNF and NF-B activation (110). NF-B can also be activated via PERK, which promotes NF-B by translational inhibition of IB via eIF2 (111). UPRER activation also occurs in macrophages, one of the primary immune cell types involved in innate immunity through phagocytosis of infectious agents. Upon exposure to pathogens, Toll-like receptors (TLRs) detect microbes to activate immune responses in macrophages. TLR2 and TLR4 specifically activate IRE1/XBP1, which are critical for sustained production of inflammatory cytokines in macrophages. IRE1 is activated upon TLR ligation via interaction with TRAF6, which promotes its phosphorylation to sustain IRE1 function (112). Mice lacking XBP1 in macrophages display increased sensitivity to infection due to impaired production of IL-6 and TNF (113). In addition to activating the IRE1/XBP1 branch of UPR, TLR activation promotes suppression of the ATF4/CHOP branch of UPR downstream of PERK. Prolonged PERK activation triggers cell death through CHOP as described above, and thus, TLRs play a critical role in suppressing ATF4/CHOP-mediated apoptosis to promote survival of macrophages (114).

Since C. elegans lack an adaptive immune system, resistance to pathogenic infection is dependent on PMK-1 (MAPK)mediated innate immunity responses, which potentially induce ER stress in the organism because of the increased secretory demand of the response (91). It has been shown that XBP-1 plays an essential role in protecting nematodes during pathogenic infection. For example, animals lacking xbp-1 exhibit major defects in ER morphology and larval lethality when exposed to Pseudomonas aeruginosa infection (115). Moreover, the increased sensitivity of xbp-1 mutants to P. aeruginosa exposure was exacerbated with simultaneous loss of pek-1 both in larval stages and during adulthood, suggesting that PEK-1 and XBP-1 function together to protect against immune activation (116). Similarly, exposure to pore-forming toxins, the most common proteinaceous exotoxin produced by bacteria, activates the IRE-1/XBP-1 pathway in a p38/MAPK-dependent manner. Loss of ire-1, xbp-1, and, to a lesser extent, atf-6 resulted in severe sensitivity of animals to pore-forming toxins (117). UPRER activation during pathogenic infection is controlled by neuronal G proteincoupled receptors (GPCRs). Specifically, the octopamine GPCR, OCTR-1, expressed in sensory neurons serves as a negative regulator of UPR, such that mutations in octr-1 increases UPR activation and promotes immunity (118, 119). Therefore, UPRER serves as a critical means to maintain ER homeostasis during pathogen infection in nematodes.

Similar to other stress responses, the innate immune response declines in function during the aging process in C. elegans. Termed immunosenescence, a decline in p38/MAPK signaling occurs during intestinal aging, allowing bacterial proliferation in the gut, which is the leading cause of death (91). As described above, promoting p38/MAPK signaling can prevent immunosenescence and extend life span independent of the UPRER. However, it is also likely that promoting canonical UPRER can promote resistance to pathogenic invasion and prevent immunosenescence. A forward genetic screen in C. elegans identified that dominant mutants of vitellogenin proteins (homologs of human apolipoprotein B-100) caused ER stress and increased sensitivity to pathogenic infection. Specifically, accumulation of mutant vitellogenins in the intestine caused collapse of the proteome and caused massive ER stress, decreasing the secretory capacity of the intestine, which is essential for mounting an efficient innate immune response. An up-regulated UPR counteracts the toxic effects of the ER stress associated with the accumulation of lipoproteins, while inhibition of UPRER via xbp-1 or ire-1 knockdown resulted in a notable increase in sensitivity to pathogens in this model (120). Moreover, another study found that overexpression of xbp-1s was sufficient to drive increased secretion of vitellogenins from the intestine, which suggests that these animals would perform better against infection (43).

The matrix of the ER is under highly oxidizing conditions in comparison to the cytosol to allow for oxidation of cysteine residues required to form intramolecular disulfide bonds during protein folding. Moreover, many enzymes that catalyze the formation of these disulfide bonds, including phosphodiesterases (PDIs), become reduced during their activity and need to be reoxidized to promote further reactions. Thus, additional enzymes, such as endoplasmic reticulum oxidoreductin 1 (ERO1), exist to provide oxidizing environments within the ER [reviewed in (121, 122)]. Ultimately, the primary functions of protein folding in the ER itself can serve as a major source of ROS and oxidative stress, especially under ER stress. Thus, under conditions of ER stress, global down-regulation of protein translation can mitigate ER oxidation and promote resistance to ER stress. In contrast, cells lacking Perk fail to down-regulate global translation through eIF2 and accumulate endogenous peroxides within the ER and experience increased oxidative stress (123).

In metazoans, the nuclear factor erythroid 2related factor 2 basic leucine zipper (NRF bZIP)family transcription factors (NRF1/2/3 in mammals and SKN-1 in C. elegans) serve to promote activation of oxidative stress defense genes. Under basal conditions, NRF2 remains in the cytosol via association with Keap1. Upon exposure to ER stress, PERK-dependent phosphorylation of NRF2 promotes NRF2 dissociation from Keap1, allowing subsequent nuclear transport and activation of NRF2 targets, including glutathione (GSH) synthesis genes responsible for buffering ROS from the ER (124, 125). While these studies highlight a clear connection between UPRER and oxidative stress response, it is unclear whether NRF2 can directly affect quality control of the ER or simply serves as a means to clear ER-induced oxidative stress. A comprehensive analysis of SKN-1 targets in C. elegans identified several UPRER targets activated directly by SKN-1. Specifically, in animals lacking functional SKN-1, ER stress failed to increase the expression of major UPRER targets, including chaperones, autophagy, calcium homeostasis, lipid homeostasis, and even UPR transcription factors themselves. Due to the failure to mount an appropriate UPRER, skn-1 mutants also exhibited increased sensitivity to multiple forms of ER stress, providing direct evidence that SKN-1 can affect ER quality control beyond its indirect roles in redox buffering (126). Perhaps most surprising in this study is that the core UPR machinery was also required for SKN-1mediated oxidative stress response. All three branches of the UPR were shown to affect skn-1 transcriptional expression, and functional IRE-1 was required for nuclear localization of SKN-1 under arsenite-induced oxidative stress (126).

Similar findings in human cells and Drosophila suggest that the integrated signaling of UPRER and oxidative stress are conserved across eukaryotes. In Drosophila, increased ER folding capacity by UPRER promotes long-term tissue homeostasis by enhancing redox response through JNK and the Nrf2 homolog CncC (127). In human HepG2 cells, NRF1 and NRF2 were shown to be required to promote the activation of ER stress signaling in response to ER stress. Specifically, NRF1 knockout cells had a diminished response to tunicamycin by ATF6, IRE1, and PERK, and partial loss of all three UPRER responses was found in NRF2 knockout cells (128).

Beyond the regulation of NRF2, UPRER components have also been shown to directly affect the transcriptional output of redox homeostasis genes. For example, ATF4 is essential for GSH synthesis to maintain redox balance of the ER (123). Moreover, XBP1 can stimulate the hexosamine biosynthesis pathway (HBP), which promotes synthesis of glycosylation products that can increase defense against ROS (129). Through these studies, it is clear that oxidative stress response and UPRER are tightly linked (Fig. 5), which begs the question of why such an extensive overlap between two distinct processes would have evolved. Perhaps the simplest explanation is that the ER serves as a major source of ROS production through its protein-folding capacity and the requirement to maintain a highly oxidative environment within its matrix, and thus, modulating NRF2 activity is critical. Beyond this, it is possible that the NRF2-UPR axis serves as a bidirectional signal between the ER and cytoplasm about its homeostatic state. As a hypothetical example, under ER stress, the UPR activates NRF2 to prepare the cytoplasm for the potential toxic effects downstream of ROS production under protein misfolding condition. Similarly, when cytoplasmic stress is high, it would be advantageous to activate a robust UPR response to promote protein folding of essential homeostatic regulators (e.g., chaperones) while also down-regulating global protein translation through eIF2.

It is becoming increasingly clear that cellular stress responses are not completely separate, and there exist notable cross communication and interdependent regulation. The UPRER and oxidative stress response (OxSR) have been shown to functionally affect the other, such that targets of XBP1s affect redox homeostasis and targets of NRF2 affect ER homeostasis. One study in C. elegans showed that transcriptional output of SKN-1 was, to a certain extent, dependent on XBP-1s function and vice versa. There are also some studies in mammalian systems that hint to similar signaling pathways, where NRF2 promotes ER quality control genes and XBP1s promotes genes involved in redox homeostasis. Another study found that glutathione synthesis genes (GSH) were potentially downstream of ATF6 signaling.

The UPRER and autophagy are two cellular processes that respond to both intra- and extracellular stressors. Both of these processes work to maintain organellar and cellular homeostasis. While it is clear that autophagy can play a role in regulating ER homeostasis by mediating lysosomal degradation of damaged ER through ER-phagy, the interplay and cross-talk between UPRER and autophagy remain poorly understood.

Autophagy is a cellular degradative process that removes damaged or unnecessary proteins and organelles to recycle macromolecules such as amino acids and lipids. Autophagy requires the coordination of more than 30 autophagy-related genes, which are involved in the formation of the autophagasome, generation of the autophagic vesicle, and fusion with the lysosome (130). Autophagy is activated under times of nutrient deprivation, mitochondrial and ER stress, cell fate and lineage decisions, and pathogen infection (131). Under conditions of ER stress, misfolded proteins accumulate in the ER and can lead to the activation of autophagy to reestablish cellular homeostasis. For example, aggregated polyglutamine in the cytosol can cause ER stressinduced activation of PERK, which induces conversion of microtubule-associated protein light chain 1 (LC1) to LC3, inducing apoptosis in an eIF2-dependent manner (132). Recent studies have shown that under conditions of ER stress, PERK can actually mobilize the major autophagy transcription factors, transcription factor EB (TFEB) and transcription factor E3 (TFE3), to translocate to the nucleus. TFEB/TFE3 activation not only leads to the induction of autophagy and lysosomal genes but also induces ATF4 and CHOP, making them more resistant to ER stressinduced apoptosis (133).

In addition, the IRE1/XBP1 pathway has been implicated in the activation of autophagy (Fig. 6). In cancer cells, XBP1s has been shown to induce autophagy through regulation of expression of Beclin2, an antiapoptotic protein, which interacts with Beclin1 to inhibit the nucleation of autophagy (134, 135). Similarly, sustained XBP1s activation in endothelial cells can promote autophagic vesicle formation, conversion of microtubule-associated protein LC1 to LC3, and expression of Beclin1. Conversely, XBP1 deficiency in mouse endothelial cells reduces LC3 expression and decreases autophagosome formation (136). IRE1 can also induce autophagy via a TRAF2-mediated pathway similar to the apoptosis machinery by inducing JNK activation and downstream Beclin1 transcription by c-Jun (137). In contrast to these studies, depletion of IRE1/XBP1 activity has also been shown to enhance autophagy and promote viability in cells obtained from patients with amyotrophic lateral sclerosis (ALS). XBP1s deficiency leads to increased forkhead Box O1 (FOXO1) expression and increased autophagy in neurons, and neuron-specific XBP1 ablation is sufficient to drive disease resistance in mice (138). These contrasting effects of the IRE1/XBP1s branch on autophagy indicate the complex interplay between the two mechanisms and highlight the importance of further research to consider targeting UPRER-autophagy cross communication as a potential avenue of therapeutic intervention.

The IRE1/XBP1 pathway has been shown to regulate autophagy both through direct transcriptional regulation of autophagic genes downstream of XBP1s and indirectly through other signaling molecules, including FOXO1 and JNK. IRE1 can promote JNK signaling through TRAF2-mediated pathways similar to the apoptosis machinery and thus activate BCL1/2 to promote autophagy. XBP1s can also activate autophagy either by inhibiting FOXO1 signaling, which releases its inhibitory effect on autophagy, or by promoting conversion of LC3 I to LC3 II.

Recent work in C. elegans has shown that activation of lysosomal activity downstream of constitutive UPRER activation via xbp-1s overexpression in neurons is crucial for xbp-1smediated longevity (139). Both cell autonomous, via intestinal xbp-1s overexpression, and cell nonautonomous, via neuronal xbp-1s overexpression, activation of UPRER induce lysosomal gene expression. In addition, xbp-1s overexpression leads to increased lysosomal activity and acidity within the intestine, which is necessary for the enhanced life span and proteostasis found in this long-lived paradigm. These processes may be mediated by HLH-30, the C. elegans homolog to mammalian TFEB, as hlh-30 knockdown is sufficient to suppress the life-span extension of neuronal xbp-1s animals. Another study has found that HPL-2, a chromatin-modifying protein, plays a critical role in ER homeostasis through autophagy. Specifically, knockdown of hpl-2 increases resistance to ER stress by promoting autophagy (140). Further, transcriptional profiling of worms deficient in phosphatidylcholine (PC) synthesis, which causes ER stress through lipid dysregulation, also induced autophagy in an IRE-1/XB-1dependent manner (141). This is highly similar to a process previously described in yeast, where inhibition of PC biosynthesis activates microlipophagy downstream of UPRER (142). These studies highlight the critical impact of UPRER on autophagy beyond canonical protein misfolding stress in the ER.

Besides the well-characterized ER chaperones and ER quality control genes, XBP1s can also transcriptionally up-regulate genes involved in N-glycan biosynthesis (143, 144) and the HBP, which generates uridine diphosphate (UDP)N-acetylglucosamine (UDP-GlcNAc), an essential substrate for both N- and O-linked glycosylation (145, 146). N-linked glycosylation begins in the ER, in which a preassembled oligosaccharide is transferred to selective asparagine residues on newly synthesized polypeptides. These oligosaccharides are essential for protein folding and maturation through the secretory pathway, and blockage of ER N-glycosylation leads to ER stress [for a detailed review, see (147, 148)]. Intriguingly, activation of XBP1s up-regulates not only genes required for ER N-glycosylation but also glycotransferases and sugar transporters in the ER and Golgi that modulate N-glycan maturation, resulting in remodeling of N-glycan structures on cell surface and secreted proteins (149). While the functional role of XBP1s-induced glycoproteome remodeling is unclear, it likely influences how cells interact with the extracellular environment and may be used to communicate ER stress between cells.

Glycosylation also regulates cytosolic and nuclear proteins via O-linked GlcNAc modifications, a dynamic posttranslational modification analogous to phosphorylation. Activation of HBP, either by XBP1s induction or by increased expression of HBP rate-limiting enzymes, enhances cellular O-GlcNAc modifications and has been shown to protect cardiomyocytes from ischemia/reperfusion injury in mice and promote proteostasis in C. elegans (145, 146). However, the specific O-GlcNAcmodified proteins that mediate such protective effects are yet to be identified. In contrast, O-GlcNAc modification on eIF2 inhibits downstream activation of UPRER, preventing ER stressinduced apoptosis (150). Additional studies will be required to understand how glycosylation changes on specific proteins during ER stress may modulate UPRER and intertissue ER stress signaling.

Peroxisomes are organelles that aid in lipid metabolism and neutralizing or using hydrogen peroxide to oxidize substrates. These functions often overlap with other cellular compartments, such as the cytosol and mitochondria, because of their overlap in metabolic processes. For example, the cytosol houses several ROS scavengers, while the mitochondria contain critical enzymes in -oxidation of fatty acids and fatty acid derivatives (87). Peroxisomes also communicate with other organelles to mediate these processes through cellular signaling pathways, vesicular trafficking, and membrane-membrane interactions. Through these complex interorganellar communications, peroxisomes regulate cellular aging in multiple ways: maintenance of the lipid bodies within the cell, exchange of metabolites between peroxisomes and other organelles, maintenance of ROS homeostasis and oxidative stress, and recycling of tricarboxylic acid cycle intermediates [refer to (151) for a more comprehensive review]. Similar to all other membrane-bound organelles, the peroxisome has a tight link with the ER, as the ER serves as the primary site for lipid and protein biogenesis of the organelle.

While there are numerous studies highlighting the importance of the ER and functional ER in maintaining peroxisomal function and biogenesis [reviewed in (152)], much less is known about the function of the peroxisome under ER stress and how UPRER affects this organelle. One study found that peroxisome deficiency can activate ER stress signaling, primarily through PERK and ATF4 signaling, which can lead to lipid dysregulation and dysfunction in cholesterol homeostasis. Specifically, peroxisome-deficient PEX2 knockout mice exhibited UPRER activation, which results in dysregulation of the endogenous sterol pathways through SREBP-2 (153). In addition, peroxisome-deficient mice showed increased peroxisome proliferatoractivated receptor (PPAR), which can cause increased expression of both SREBP-2 and the transcriptional regulator p8, leading to increased ER stress. Sustained p8 and UPRER activity can contribute to the development of hepatocarcinogenesis (154). Despite these studies highlighting a link between ER and peroxisomes, it is still unclear how peroxisome dysfunction leads to ER stress. Are the effects simply indirect where lipid dysregulation upon peroxisome dysfunction leads to ER stress? Or is there a causative link between ER and peroxisome function?

The current state of the literature has made it evident that the ER serves numerous critical functions outside of protein homeostasis. As such, the quality control machineries dedicated to preserving ER form and function, such as the UPRER, are essential in homeostatic regulation of these alternative functions, including lipid metabolism, autophagy, apoptosis, redox homeostasis, and glycosylation. Here, we briefly discussed how the UPRER affects these other functional roles of the ER independently. However, a critical question is how these functional roles overlap and whether the homeostatic regulation of these pathways can be separated. It is clear that when the UPRER is activated, many downstream targets are simultaneously regulated. For example, under conditions of protein misfolding stress, lipid homeostasis genes downstream of IRE1/XBP1 are activated in addition to chaperones and protein repair machinery. Thus, is it sufficient to promote a single component downstream of UPRER, or is it essential to simultaneously maintain all functions of the UPRER? Alternatively, if lipid homeostasis of the ER is enhanced in the absence of protein quality control machinery, would that be detrimental? Is there an essential balancing act that occurs between all the functional roles of the ER? And if so, how does the cell modulate this balance?

Beyond the beneficial roles of the UPRER, we also discussed how sustained and unresolved UPRER signaling can be detrimental. However, often the detrimental effects of the UPRER are described under conditions where there is unresolved ER stress. Hyperactivation of the UPRER in the absence of stress is generally a beneficial phenomenon and promotes metabolism, organismal health, and life span [reviewed in (6)]. Note that there do exist some specific circumstances where even UPRER hyperactivation in the absence of stress can also be detrimental. For example, overexpression of xbp-1s in the muscle of C. elegans decreases life span (14), and overexpression of HAC1s (the S. cerevisiae homolog of XBP1) can perturb cell cycle progression (155). Therefore, how does a cell differentiate between a beneficial and detrimental UPRER signature? Do there exist other transcriptional regulators that function with canonical UPRER transcription factors to alter the downstream signaling cascade? We briefly discussed the interplay between SKN-1 and XBP-1 in C. elegans. What are the other transcriptional cofactors of the canonical UPRER transcription factors, and how do they serve as sensors to inform the cell of when UPRER activation is beneficial or damaging?

An additional concern in studying quality control mechanisms is that, historically, research is generally focused on a single, organelle-specific machinery. However, current research has made it apparent that communication between homeostatic and stress response machineries is not only common but also critical. For example, we described the complex interplay between the oxidative stress response and the UPRER that is impossible to disconnect. Moreover, as the ER is not the only organelle responsible for producing ROS, it comes as no surprise that mitochondrial quality control machineries are also highly interconnected to the oxidative stress response (156). How then do all these quality control machineries communicate with one another? Under conditions of competing needs, such as through general stress where several organelles are damaged, which stress response pathway is preferentially activated? Can all cellular stress responses be mutually activated in a way that is beneficial to the cell? Hyperactivation of a single stress response is generally sufficient to promote organismal healthspan and life span [reviewed in (1)]. In these models, is it possible that other quality control machineries are also activated? Or would hyperactivating multiple stress response pathways simultaneously have a compounded effect and create a super long-lived organism? Conversely, is it possible that hyperactivating too many stress response pathways would be detrimental for an organism?

Last, we still know relatively little about cross communication of the stress signals identified here across cell and tissue types. While cell nonautonomous signaling has generally been heavily studied in the realm of the UPRER, most of these studies focused primarily on the canonical role of the UPRER in protein homeostasis. Very recent studies have now emerged in how nonautonomous communication of UPRER from the nervous system to the periphery can promote lipid homeostasis in distal tissues, as described above. Even in these studies, the actual signaling events that happen across tissues are still poorly understood. Do there exist similar cell-to-cell communication events for regulation of autophagy, immunity, oxidative stress response, etc.? If so, are the signaling molecules and receptors involved similar to or distinct from those already identified? Answering these questions described above is critical in furthering our understanding of the impacts of manipulating the UPRER for therapeutic intervention. Because of the pleiotropic effects of the UPRER described here, it is clear that targeting the master regulators of UPRER activation is unwise. However, downstream targets of UPRER can be targeted for specific diseases, ideally in specific tissue types of interest.

Acknowledgments: We would like to thank all members of the Dillin laboratory for feedback and technical/scientific support, with special thanks to R. Bar-Ziv and A. Frakes for careful review of the manuscript. Funding: M.G.M. was supported by 1F31AG060660-01 through the National Institute of Aging (NIA), R.H.-S. was supported by the Glenn Foundation for Aging Postdoctoral Fellowship and grant 1K99AG065200-01A1 from the NIA, and A.D. was supported by 4R01AG042679-04 through the NIA and the Howard Hughes Medical Institute. Author contributions: M.G.M. prepared all figures and wrote the autophagy and peroxisome sections. R.H.-S. wrote the abstract, introduction, apoptosis, immunity, oxidative stress response, and concluding remarks sections. G.G. and R.H.-S. wrote the lipid homeostasis section. C.K.T. wrote the glycosylation section. A.D. provided intellectual contributions. All authors edited the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: No data were produced in this manuscript.

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Clare Boothe Luce Scholarship Prepares Women Scientists for the Future – St John’s University News

July 15, 2020

Named for the visionary woman who excelled in myriad fields, St. Johns Clare Boothe Luce (CBL) Undergraduate Scholarship encourages gifted women to pursue collegiate studies in the sciences and technologyareas in which women historically are underrepresented. The CBL scholarship is an outgrowth of the UniversitysWomen in Science (WIS) Scholarship Program. St. Johns College of Liberal Arts and Sciences Class of 2020 included three recipients of the prestigious scholarship, each with an inspiring story to tell.

When toxicology major Kathryn Bozell enrolled at St. Johns four years ago, she was not even aware of the CBL scholarship. This fall, the scholarship recipient returns to the University to pursue her masters degree in toxicology as a CBL Graduate Fellow.

During her first year, Kathryn, a native of Louisville, KY, was encouraged to apply for the CBL scholarship by several faculty, who saw great promise in the budding scientist.

Upon learning about the exciting opportunities available through WIS and the CBL scholarship program, I eagerly applied, she recalled. The scholarship program provided me with the opportunity to connect with incredible female mentors and peers. It also inspired me to continue my studies and pursue a masters degree.

The experience also served as a launchpad for her research on the effects of copper dimethyldithiocarbamate (CDDC) on the release of a protein that is known to propagate the inflammation of nervous tissue. Neuronalinflammation has been linked to neurodegenerative diseases, such as Alzheimers disease, Parkinsons disease, and Multiple Sclerosis.

Participating in WIS activities was an integral part of my academic and professional development at St. Johns, she recalled. In addition to the invaluable networking opportunities it offered, it provided me the chance to build personal relationships with other women in science, which greatly enriched my academic experience overall.

Kathryn is excited to return to campus to begin her graduate work and serve as a role model for younger students. As I continue my education and research, I am excited to inspire the next generation of women in science in the same manner, she said. I would highly encourage all young women interested in a career in the sciences to learn more about the Clare Boothe Luce Scholarship program.

For Teagan Sweet, the CBL scholarship was a connection to a welcoming community of female scientists at St. Johns and around the globe.

CBL was such a pivotal experience for me, the native of North Attleborough, MA, recalled. I loved being surrounded byand supported bythe strong women in STEM at St. Johns who became my role models. CBL validated my experience in science.

That experience saw the chemistry major complement her study of the field with minors in photography and international studies. She also explored computational research, focusing on understanding how orientation and the folding of proteins leadto large-scale changes in the cell.

Teagan traveled to Dublin, Irelands Trinity College to work on the development of new green materials, which could one day lead to advances in energy storage, solar cells, and drug delivery.

In addition to her rigorous course load, Teagan was Head Skull of the Skull and Circle Honor Society, St. Johns Colleges highest honor for students, and was awarded the prestigiousJeannette K. Watson Fellowshipa three-year, international internship program funded by the Thomas J. Watson Foundation. She was also an S-STEM scholar and contributed to research in collaboration with the National Science Foundation, which focused on the development of a biodegradable water filter to be used in disaster situations.

Both the WIS and CBL programs assisted Teagan in her graduate school application process, through mentorship, as well as words of wisdom. This fall, she will pursue a Ph.D. in inorganic chemistry at the University of Notre Dame.

Thanks to these programs, I feel especially connected not only to women in STEM at St. Johns, but across the country, as well, she said. I will always be proud to be a part of this elite, intelligent community.

Like many students, Natalie Williams entered her senior year unsure of her postgraduation plans. A chemistry major with a minor in graphic design, Natalie sought the advice of a faculty mentor, who suggested she pursue a career where she could combine her passion for chemistry with her love of the arts.

One of my professors told me about science-related research at art museums, she recalled. I had not given this field any serious thought, but now my goal is to be a scientific researcher at a museum.

In pursuit of that goal, Natalie will attend Yale University this fall, where she will work toward her Ph.D. in material chemistry. While her focus now is on the future, she looks back on her four years at St. Johns with fondness and gratitude.

The CBL scholarship helped me not only financially, but professionally, making the way for new and lasting professional connections, she said. This program introduced me to fellow women in science who will always serve as my inspiration.

Natalie was a member of St. Johns National Science Foundation-funded S-STEM Scholars Program, which introduced her to undergraduate student research, including a research group in the chemistry department that designed, synthesized, and analyzed materials using DNA nanotechnology. There, she was able to combine her chemistry and biotechnology skills with her graphic design knowledge and made nanometer-scale DNA origami objects, which fold themselves into particular shapes.

She was also a member of the American Chemical Societys Scholar Program, an extremely competitive program for underrepresented minority students who plan to pursue careers in chemistry. In addition, Natalie participated in the BIOMOD research competition, an international bio-molecular design competition for students sponsored by the Wyss Institute for Biologically Inspired Engineering at Harvard University.

Natalie is grateful for the support she received as a CBL scholar as St. Johns. Everyone here gave me great advice that helped guide me in the best direction to achieve my goals, she recalled. Conducting research on art is something that truly fascinates me, and I plan to fulfill this dream.

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Arizona universities join forces to contribute to COVID-19 modeling and simulation efforts – ASU Now

July 14, 2020

While the COVID-19 pandemic continues to impact the world, the research computing centers at Arizona State University, Northern Arizona Universityand University of Arizonahave united as a team to contribute to the Folding@homeproject. The project utilizes idle computing power to significantly contribute to vital scientific research and therapeutic drug discovery.

The Arizona Research Computing consortium is contributing to this collective effort by using advanced computing resources to perform complex protein modeling computations during brief idle periods on local supercomputers. By running these computations only during downtime, contributions to COVID-19 modeling and simulation efforts can be made through the Folding@home project without impacting everyday research. Artist rendition of the SARS-Cov-2 virus. The envelope protein is shown in cyan. (Figure from Klick health https://covid19.klick.com/) Download Full Image

The Folding@home project provides people around the world the opportunity to make active contributions to a variety of scientific research efforts including COVID-19. Volunteers, or citizen scientists, can download the Folding@home software to their personal computers, allowing simulations of complex scientific processes to run in the background while their personal computers are not in use. The Folding@home project is crowdsourcing at its best, using shared computing power at a massive scale to help solve grand challenges in biomedical research.

In addition to mobilizing citizen scientists across the globe, many institutions and corporations are contributing their own computational resources such as high performance workstations and servers. This distributed computational power is estimated to be 10 times faster than the worlds fastest individual supercomputer.

The onslaught of COVID-19 has raised the visibility of the Folding@home project, highlighting a unique opportunity to fight the virus. The project seeks to understand how proteins, which are large, complex molecules that play an important role in how our bodies function, fold to perform their biological functions. This helps researchers understand diseases that result from protein misfolding and identify novel ways to develop new drug therapies.

How proteins fold or misfold can help us understand what causes diseases like cancer, Alzheimer's disease and diabetes. It might also lend insight into viruses such as SARS-CoV-2,the cause of the recent COVID-19pandemic.

Imagine if I told 100 people to fold a pipe cleaner. They are going to fold it in 100 different ways because theres an infinite number of combinations of how to take something that is straight and fold it," said Blake Joyce, assistant director of research computing at the University of Arizona."Thats what viruses and living things do with proteins. They make copies of themselves and fold them up in their own particular way.

Using computational modeling, researchers can explore the mechanics of proteins of the virus and predict every possible way it might fold, or physically change shape.

In biology, shape is function. If you can disrupt that shape, the virus is inactive or cant do its thing. If you disrupt any of the mechanisms that can damage us, you have a cure, or at least something you can treat. And that is what were after. It just takes a lot of computing to come up with every possible way to bend a pipe cleaner, Joyce said.

By running computer simulations, researchers can take the virus and see how it interacts with various compounds or drugs and narrow down which ones might work to interrupt one of the critical mechanisms the virus needs to survive.

Folding@home assigns pieces of a protein simulation to each computer and the results are returned to create an overall simulation. Folding@home computations for COVID-19 research seem to be most productive on the kind of computers found in facilities like Arizonas research computing centers, making their contributions even more valuable.

Volunteers can track their contributions on the Folding@home website and combine their efforts as a team, receiving points for completing work assigned to them and even earning bonus points for work that is more computationally demanding or that might have a greater scientific priority.

The Arizona Research Computing team has risen quickly in the ranks, highlighting the powerful computing capabilities at Arizonas state universities and the effectiveness of regional collaborations. As of mid-June, the Arizona Research Computing team was ranked in the top 100 out of nearly a quarter of a million teams, surpassing Hewlett Packard, Cisco Systems, Apple Computer, Inc., Google, Ireland and Poland, as well as many other university, industry and national or international contributors.

The Folding@home project investigates many research questions that require an enormous amount of computing, but this specific use for COVID-19 provides a unique opportunity, spurring many computing centers to participate in Folding@home for the first time, said Gil Speyer, lead scientific software engineer for Arizona State Universitys research computing center.

Todays biomedical research requires vast amounts of time and computing power. While the Arizona Research Computing team may directly impact COVID-19 research in a small way, the overall impact of the Folding@home project is much broader and will continue to have applications beyond the COVID-19 pandemic.

ASU:

Sean Dudley, assistant vice president and chief research information officer, Research Technology Office

Douglas Jennewein, senior director, research computing, Research Technology Office

Gil Speyer, lead scientific software engineer, Research Technology Office

Marisa Brazil, associate director, research computing, Research Technology Office

Jason Yalim, postdoc,research computing, Research Technology Office

Lee Reynolds, systems analyst principal, research computing, Research Technology Office

Eric Tannenhill, senior software engineer,research computing, Research Technology Office

NAU:

Chris Coffey

UArizona:

Blake Joyce, assistant director, research computing

Todd Merritt, information technology manager, principal

Ric Anderson, systems administrator, principal

Chris Reidy, systems administrator, principal

Adam Michel, systems administrator, principal

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Learn the ‘right way’ to make scrambled eggs with this French technique – TODAY

Scrambled eggs are a breakfast staple and everyone seems to have their own particular method for cooking up the fluffiest eggs possible. But the quest for the perfect scrambled eggs may be over, thanks to this French-style recipe by private chef and YouTuber Bruno Albouze.

Unlike traditional scrambled eggs, which are fluffy and form more solid curds, the French style offers a silkier, creamier variation on the breakfast food, Albouze told TODAY.

If (the eggs are) cooked the right way, it changes the texture, the mouthfeel is just incredible, he said. If it's overcooked, you just don't get the same thing. You're pretty much eating chopped omelet.

According to Albouze, the French way of cooking scrambled eggs is the right way.

While Albouze may have mastered this classic technique, he didnt invent it. Chef Brendan Walsh, the dean of the School of Culinary Arts at The Culinary Institute of America, explained that while scrambled eggs have likely been around for thousands of years in places where chickens were first domesticated (like China and Egypt), the French style emerged in the early days of haute or "high cuisine." This term refers to the style of food preparation that blossomed in France during the 16th century, and is still served in many Michelin-starred eateries today.

Eggs are a staple in cooking school," Albouze said. "Cooking eggs is a big thing, because eggs are the trickiest thing to cook. It's composed mostly of water, because the egg white is watery, and the yolk is fat, but it's tricky because it can overcook very fast.

"Scrambled eggs are definitely the item not everyone knows how to cook it properly. And very very few restaurants know how to do it without them being overcooked.

Albouze, 50, has been cooking since he began an apprenticeship at the age of 14 in France, where he learned the art of cooking, baking and pastry making. Growing up, Albouze said he used to walk to nearby farms for fresh, pasture-raised eggs to make omelets and scrambled eggs.

During his career, the chef has worked at the Htel Plaza Athne in Paris under renowned chef Alain Ducasse and as an instructor at the Culinary Institute Lenotre in Houston.

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But the chef, who now lives in San Diego, really found his stride in 2009 when he started his own YouTube channel. Albouze now has over 760,000 subscribers and 70 million total video views. One of his most popular videos, a recipe for ratatouille, has garnered over 10 million views.

In one of his latest cooking demos, Albouze shared his favorite method for cooking scrambled eggs in the French style. In 2015, he showcased how to cook French-style eggs in water bath. Both methods require constant stirring and movement over low heat to achieve the desired creamy texture.

One method Albouze demonstrated uses a nonstick pan, while the other uses a water bath, which is known as a "bain marie in French and culinary terms.

Walsh added that the French way is a technique where "slow and low, patience and respect" result in the creamiest eggs possible. It is a preparation method that is taught at the CIA, although he said he rarely sees the technique used in America outside of fancier hotels and restaurants.

"It is important in egg cookery to create soft and supple textures," Walsh said via email. "Too high of heat will dry out the protein and take away the supple creaminess that the protein can provide.

Cooking is about ingredients, Albouze added. 80% (is) about the ingredients, 10% skills and 10% time. So, it tells us you don't have to be skilled, just if you understand that ingredients are the most important part of cooking.

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For this reason, he recommends using pasture-raised eggs, which means that the egg-laying hens are allowed to spend plenty of time outside, and feed on grass, bugs or worms instead of a corn- or soy-based diet.

These days, if the egg aisle at your supermarket seems a little empty, it might not be a bad idea to take a road trip out to a local farm, pick up some fresh eggs and try out this fun technique at home.

Said Walsh, Crispy toast and creamy French-style eggs are just the sexiest start to the chefs morning."

This recipes serves two people and takes about 15 minutes from start to finish.

Said Albouze, There's no question that the most important factor when cooking eggs is the cooking technique itself. In the case of scrambled eggs, that means using gentle heat and taking the eggs off the flame a little early to account for carryover cooking.

1. Crack eggs into a bowl and set aside. Do not whisk the eggs. Set flame to medium and allow heat to gently warm the pan prior to adding any fat.

2. Add the olive oil and butter to the pan. Let melt slightly before adding the unbeaten eggs. With a whisk or a rubber spatula, poke the egg yolks and cook slowly on medium heat while constantly stirring, folding and shaking the pan.

3. Continue to cook and move the eggs as they start to scramble. They should yield a soft, delicate and creamy texture in about 5 minutes. As the eggs start to come together, turn off the heat and continue to stir the eggs.

4. To stop the cooking process, whisk in a dash of milk or cream. Season the eggs with salt and a few grinds of pepper before youre ready to serve. Salting the eggs too early may make your eggs watery.

5. If desired, serve the scrambled eggs with some fresh herbs, such as dill or chives. Eggs also pair remarkably well with caviar, salmon roe, truffle, cured salmon or bacon. Either way, these eggs should be served immediately.

Bon apptit!

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Tennessee scientists have partnered on cutting-edge discoveries in a race against COVID-19 – Knoxville News Sentinel

Scientists at Oak Ridge National Laboratory havetaken an important step in the race to understand SARS-CoV, the virus that causes COVID-19. Using sophisticated techniques, the scientists mapped out the structure of a critical protein of the coronavirus.

They'rehoping to answer this age-old scientific question that's more pressing than ever in this pandemic:How do you kill something that's not really alive?

The results of the groundbreaking work at Oak Ridge National Laboratory were published in Nature,a leading science journal.

Without this protein, the coronavirus cannot replicate. The scientists hope that by studying this structure, they will be able to find drugs that can stop it.

"The most important part is probably the fact that this protein is essential for the replication of this virus," said Dr.Daniel Kneller, a researcher at Oak Ridge National Lab andthe first author of the study. "If you inhibit this protein you're preventing the virus from assembling. Period."

While this study is focused on a very specific aspect of COVID-19, it opens a window onto the immense network of scientists and scientific organizations working on the pandemic. Tennessee is home to a state-spanning, drug development pipeline that is a microcosm of nationaland global research.

The COVID-19 protease protein is both shaped like a heart and functions as one. Without it the virus cannot grow and spread.(Photo: Andrey Kovalevsky, ORNL, U.S. Department of Energy)

Viruses are hard to treat because they arent like other things that cause diseases. Antibiotics, antifungals and antiparasitic drugs stop cells from replicating or kill them outright. But viruses arent made of cells. They are bundles of proteins and genetic material that hijack cells, forcing them to produce viral particles. Viruses cannot replicate on their own.

Theres debate among biologists as to whether viruses are actually alive because of this.

"How do you kill something that's not really alive?" said Dr. Martha S. Head, director of the Joint Institute for Biological Sciences at Oak Ridge National Lab. She oversees Oak Ridge's COVID-19 molecular design research projects. She explained that this study was part of a push to shut down viral replication at multiple stages, which is how HIV is treated.

"That combination (of drugs) shuts down so many parts of the (HIV) life cycle that you drive down viral loads to where they don't matter," Head explained.

COVID-19 Protease crystals, grown in Oak Ridge National Labs Protein Crystallization and Characterization laboratory and pictured in microscopic view.(Photo: Daniel Kneller: ORNL, U.S. Department of Energy)

To do this, the Oak Ridge team went right to the heart of the coronavirus itsproteins. When a virus infects a cell, it forces the cell to produce viral proteins. But host cells often cant create finished viral proteins, just long strands of unfinished, conjoined, protein.

Proteins are a bit like self-folding origami. Once theyre assembledthey fold into their final shape. Some proteins need to be cut by enzymes, like a protease, to get them into their final shape.Viral proteins cant do that when they're conjoined.

To make finished protein, each viral particle carries a protein enzyme called protease. The COVID-19 protease cuts unfinished viral protein, freeing it to fold itself into a final shape. If the protease cant do this, new virus cannot be made.

It is the heart of viral replication. Finding a chemical compound that can attach to and stop the heart of COVID-19 could be critical for developing a treatment. This is actually the therapeutic approach for anti-HIV drugs like Atazanavir.

But to quickly discover a drug,scientists it helps to have anaccurate map of the protease.

"If part of the protein is incorrectly modeled when you try to design drugs you may miss interactions that would otherwise form (between the drug and the protein)," explained Dr. Andrey Kovalesky, a researcher at Oak Ridge who worked on the study. He explained that without an accurate structure model, researchers might miss a potential drug or get bogged down in false positives.

To find the structure, the scientists grew large crystals made of viral protein in the lab. They took these crystals and exposed them to x-rays. When x-rays hit the protein crystal, they bend and scatter in different directions based on the shape of the protein. After they scatter they hit sensitive x-ray detectors. The scientists use the pattern of x-ray hits to ultimately figure out the shape of the protein.

This is called x-ray crystallography and was famously used to discover the structure of DNA.

The technique is like taking multiple photographs from different angles of the same object. By looking at all angles of the object you can figure out its 3-D structure.

Usually this kind of experiment is done at very cold temperatures. Thats because protein molecules tend to move more at warm temperatures. Its like photographing a moving object.

Unfortunately, getting a clearer picture can also mean missing theshape of the protein or how they move. Viruses often change shape but they cant when theyre frozen. Its like looking at frozen meat and expecting it to behave like a living muscle.

"You really have to appreciate that it's one single confirmation that you're looking at," said Dr. Paul McGonigle,director of the Drug Discovery and Development Program at Drexel University. "You hope that this is the confirmation the protein exists in most of the time, but you never know for sure."

The scientists at Oak Ridge did something special. They did this study at room temperature. Their equipment is more sensitive than the type typically used for this kind of experiment. Because of that, they could see the a fuller range of motion in the of COVID-19 protein that accuracy is very important for developing a drug.

"I think it's useful for them to have these different confirmations to target," said Dr. Ole Mortensen, associate professor of pharmacology at Drexel University. Mortensen explained that his own drug development work was made more challenging because he only had a single snapshot of his target protein.

"I'm worried that I could be missing some of the other ones. I think it makes sense what they're doing. They're opening up more possibilities." Mortensen said.

Oak Ridge might not immediately come to mind when you think medical research. But the national lab system has played a role in medical science since its inception. Sex chromosomes were discovered by pioneering geneticistLiane Russell at Oak Ridge, for example.

When Congress injected hundreds of millions of dollars into COVID-19 research through the CARES Act, The Department of Energy received $99.5 million for the national lab system. Compared to other agencies like the National Institutes of Health or Department of Defense, which got $945 million and $415 million respectively, that might not seem like a lot.

But the national lab system has unique resources that can be quickly marshaled against COVID-19. The Neutron Spallation Facility has the kind of sensitive x-ray detectors necessary to scan a protein at room temperature. The facility houses a lab capable of quickly growing large protein crystals.

"Oak Ridge is uniquely good at growing really big protein crystals," said Charles Sanders, a professor of biochemistry at Vanderbilt University. "Because they have that general expertise, it lets them do room temperature crystallography."

"They also have a network of people around the country, so if the big dogs at these agencies want stuff to happen then it can be, a wartime response, basically," Sanders said.

When the CARES act passed, it let the scientists clear their schedule and focus on COVID-19. Ordinarily, research like this takes months if not years of applying for grants and negotiating for time on equipment. This study mapped and published the protease structure in about a month.

"This is different than our usual projects," said Dr. Head. "Acrossthe Department of Energy as a whole, the speed (of organizing research) is astronomically fast."

Importantly, Oak Ridge houses the Summit supercomputer, one of the fastest supercomputers in the world. Once the structure was figured out by one team, the Summit team quickly screened it against a massive library of potential compounds, looking for potential matches.

"We broke a world record on the supercomputer," said Jeremy Smith,director of the Center for Molecular Biophysics at Oak Ridge. "We screened 1.2 billion compounds in less than a single day."

This is not the first time Dr. Smith has run a massive simulated drug test like this. Knox News covered his experiments back in March. The difference here is scale. Smiths team screened the COVID-19 protease against 1.2 billion possible drugs in a single day using Summit's whole processing system. Now the most promising candidates are being sorted out for eventual testing against live COVID-19 virus.

As impressive as Oak Ridges facilities are, they dont have the ability to do that kind of testing in house. For that, Smith turned to Dr. ColleenJonsson, a professorUniversity of Tennessee Health Sciences Center in Memphis.

Dr. Jonsson is an experienced virologist and virus hunter. She also happens to be the director of the Southeast Regional Biocontainment Laboratory, one of a small network of labs authorized by the federal government to do research on dangerous biological agents and emerging infectious diseases. She had the facility and staff to do what Oak Ridge could not:validate potential drug targets in the real world.

"A virtual screen (in a computer)is a theoretical screen." Dr. Jonsson said. "Once we find them we have to validate we're actually hitting the right thing."

Jonsson saidthat earlier in the year Smith reached out to her to test possible drugs targeting a different protein, the spike protein the coronavirus uses to attach to cells. Since then, her lab has expanded to validating other potential drugs targeting other proteins. While Dr. Jonsson hasnt yet begun to work on the COVID-19 protease, she expects to shortly.

This part of the process, where drugs are tested in live cells to see if they stop a virus from replicating, is arduous. Scientists working at the Biocontainment Lab don full biohazard suits to run their tests. Even after they validate that a potential drug works on a virus in a dish, they still need to run extensive dosage and safety testing, a process that can take months if not years.

Then they have to see if the drug actually works in a real infection. For this they need to test the drug in an infected animal that gets infected with COVID-19 like a human would. Getting an accurate animal model is a difficult process that requires its own experiments and validation.

Dr. Jonsson's team has been busy with this, and other COVID-19 work, since the start of the pandemic. They are among the very few people reporting to work at the University of Tennessee Health Sciences Center campus during the initial lockdowns. The Biocontainment team worked quickly to get everything ready for coronavirus research.

"They worked every day through the Safer at Home order with remarkable dedication," said Dr. Jonsson. "Everyone was working seven days a week to get everything ready."

In spite of its critical role in coronavirus research, the Biocontainment Lab is operating semi-independently. It has not received any funding yet through the CARES Act and is working solely on University of Tennessee funding.

None of thisscience is settled. The author of another structural study on the COVID-19 protease,Dr. Rolf Hilgenfeld of the German Center for Infection Research, was not convinced that the Oak Ridge study would amount to anything.

"I don't think this small difference, (between the shape of the proteins at different temperatures)whatever is its cause, matters for drug design," wrote Hilgenfeld in an email to Knox News.

The Oak Ridge team is planning to scan COVID-19 proteins using a higher resolution technique, neutron scattering,to get even better structures. Dr. Jonsson's team hopes to be running animal tests for possible COVID-19 drugs by the end of the year.

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Tennessee scientists have partnered on cutting-edge discoveries in a race against COVID-19 - Knoxville News Sentinel

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