Category Archives: Protein Folding

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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

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

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

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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In terms of geography, the U.S. and Canada holds major market share of treatments for syndromes of progressive ataxia and weakness disorders market in North America. In Europe, Germany, France and the U.K are major markets for treatments of syndromes of progressive ataxia and weakness disorders.

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.

However, patent expiries of major drugs hampers growth of the treatments for syndromes of progressive ataxia and weakness disorders market. Moreover, stringent regulations and standard requires for approval process of new drugs impede growth of the treatments for syndromes of progressive ataxia and weakness disorders market. Several government agencies, such as FDA and European Medicines Agency, are responsible for the approval of every drug. In addition, the approval process takes a very long time to approve a specific drug.

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Some of the major companies operating in the treatment for syndromes of progressive ataxia and weakness disorders market are Abbott Laboratories, Acorda Therapeutics Inc., American Regent Inc., Baxter International Inc., Biogen Idec., Bristol-Myers Squibb, Cadila Healthcare Ltd., Eli Lilly and Company, Glaxosmilthkline Plc., Sanofi, Roche Holding Ltd., Pfizer Inc. and Novartis AG.

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

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.

Original post:
Beyond the cell factory: Homeostatic regulation of and by the UPRER - Science Advances

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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Clare Boothe Luce Scholarship Prepares Women Scientists for the Future - St John's University News