RESUMEN
Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Imagenología Tridimensional , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Escherichia coli/enzimología , Escherichia coli/metabolismo , Espectrometría de Masas , Simulación de Dinámica Molecular , Presión Osmótica , Fosforilación , Proteolisis , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Estrés FisiológicoRESUMEN
Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells, but our knowledge of the metabolite-protein interactome lags behind our understanding of protein-protein or protein-DNA interactomes. Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environment. The approach identified a network of known and novel interactions and binding sites in Escherichia coli, and we demonstrated the functional relevance of a number of newly identified interactions. Our data enabled identification of new enzyme-substrate relationships and cases of metabolite-induced remodeling of protein complexes. Our metabolite-protein interactome consists of 1,678 interactions and 7,345 putative binding sites. Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.
Asunto(s)
Metaboloma , Proteoma/metabolismo , Proteómica/métodos , Transducción de Señal , Programas Informáticos , Regulación Alostérica , Sitios de Unión , Escherichia coli , Metabolómica/métodos , Unión Proteica , Mapas de Interacción de Proteínas , Proteoma/química , Saccharomyces cerevisiae , Análisis de Secuencia de Proteína/métodosRESUMEN
Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.
Asunto(s)
Arginina/metabolismo , Linfocitos T CD4-Positivos/inmunología , Inmunomodulación , Activación de Linfocitos , Melanoma Experimental/inmunología , Neoplasias Cutáneas/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al ADN/metabolismo , Técnicas de Inactivación de Genes , Glucólisis , Humanos , Memoria Inmunológica , Metaboloma , Ratones , Ratones Endogámicos BALB C , Fosforilación Oxidativa , Proteoma , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.
Asunto(s)
Bases de Datos de Proteínas , Proteoma , Acceso a la Información , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Colesterol/biosíntesis , Docetaxel , Femenino , Humanos , Internet , Hígado/efectos de los fármacos , Masculino , Mutación , Neoplasias de la Próstata/tratamiento farmacológico , Empalme del ARN , Taxoides/uso terapéuticoRESUMEN
Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.
Asunto(s)
Senescencia Celular , Longevidad , Longevidad/genética , Polimerizacion , AminoácidosRESUMEN
Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges-crossovers-between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.
Asunto(s)
Intercambio Genético/fisiología , Meiosis/fisiología , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Espectrometría de Masas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.
Asunto(s)
Escherichia coli , Estabilidad Proteica , Proteoma , Proteoma/metabolismo , Proteoma/química , Humanos , Escherichia coli/metabolismo , Temperatura , Betaína/química , Betaína/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Trehalosa/química , Trehalosa/metabolismo , Proteómica/métodos , Prolina/química , Prolina/metabolismo , Glucosa/química , Glucosa/metabolismo , Glicerol/química , Glicerol/metabolismo , MetilaminasRESUMEN
Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gßγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.
Asunto(s)
Adenilil Ciclasas , Calmodulina , Animales , Bovinos , Adenilil Ciclasas/química , Adenilil Ciclasas/metabolismo , Colforsina/farmacología , Microscopía por Crioelectrón , Proteómica , Proteínas de Unión al GTP/metabolismoRESUMEN
Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.
Asunto(s)
Calmodulina , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Calmodulina/metabolismo , Activación del Canal Iónico/fisiología , Microscopía por Crioelectrón , Calcio/metabolismo , Nucleótidos Cíclicos/farmacología , GMP Cíclico/metabolismoRESUMEN
For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Proteínas , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , ProteolisisRESUMEN
The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis-mass spectrometry (LiP-MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP-MS detects well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson's disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.
Asunto(s)
alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , Mapeo de Interacción de Proteínas , Espectrometría de Masas , Unión Proteica , Proteolisis , Enfermedad de Parkinson/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Mapas de Interacción de Proteínas , Conformación Proteica , Amiloide/metabolismo , Amiloide/química , Proteoma/metabolismoRESUMEN
The complexity of many cellular and organismal traits results from the integration of genetic and environmental factors via molecular networks. Network structure and effect propagation are best understood at the level of functional modules, but so far, no concept has been established to include the global network state. Here, we show when and how genetic perturbations lead to molecular changes that are confined to small parts of a network versus when they lead to modulation of network states. Integrating multi-omics profiling of genetically heterogeneous budding and fission yeast strains with an array of cellular traits identified a central state transition of the yeast molecular network that is related to PKA and TOR (PT) signaling. Genetic variants affecting this PT state globally shifted the molecular network along a single-dimensional axis, thereby modulating processes including energy and amino acid metabolism, transcription, translation, cell cycle control, and cellular stress response. We propose that genetic effects can propagate through large parts of molecular networks because of the functional requirement to centrally coordinate the activity of fundamental cellular processes.
Asunto(s)
Herencia Multifactorial , Proteínas de Saccharomyces cerevisiae , Transducción de Señal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , FenotipoRESUMEN
Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from CRISPR interference (CRISPRi) with 352 genes in all major essential biological processes. Next, on the basis of the comparison of genetic changes with 1,342 drug-induced metabolic changes, we made de novo predictions of compound functionality and revealed antibacterials with unconventional modes of action (MoAs). We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality from bacteria to human cell lines.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Metabolómica/métodosRESUMEN
The rise of systems biology implied a growing demand for highly sensitive techniques for the fast and consistent detection and quantification of target sets of proteins across multiple samples. This is only partly achieved by classical mass spectrometry or affinity-based methods. We applied a targeted proteomics approach based on selected reaction monitoring (SRM) to detect and quantify proteins expressed to a concentration below 50 copies/cell in total S. cerevisiae digests. The detection range can be extended to single-digit copies/cell and to proteins undetected by classical methods. We illustrate the power of the technique by the consistent and fast measurement of a network of proteins spanning the entire abundance range over a growth time course of S. cerevisiae transiting through a series of metabolic phases. We therefore demonstrate the potential of SRM-based proteomics to provide assays for the measurement of any set of proteins of interest in yeast at high-throughput and quantitative accuracy.
Asunto(s)
Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/química , Espectrometría de Masas/métodosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Cellular behavior is controlled by the interplay of diverse biomolecules. Most experimental methods, however, can only monitor a single molecule class or reaction type at a time. We developed an in vitro nuclear magnetic resonance spectroscopy (NMR) approach, which permitted dynamic quantification of an entire 'heterotypic' network-simultaneously monitoring three distinct molecule classes (metabolites, proteins and RNA) and all elementary reaction types (bimolecular interactions, catalysis, unimolecular changes). Focusing on an eight-reaction co-transcriptional RNA folding network, in a single sample we recorded over 35 time points with over 170 observables each, and accurately determined five core reaction constants in multiplex. This reconstruction revealed unexpected cross-talk between the different reactions. We further observed dynamic phase-separation in a system of five distinct RNA-binding domains in the course of the RNA transcription reaction. Our Systems NMR approach provides a deeper understanding of biological network dynamics by combining the dynamic resolution of biochemical assays and the multiplexing ability of 'omics'.
Asunto(s)
Redes Reguladoras de Genes , Metaboloma , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/análisis , ARN/análisis , Células HEK293 , Humanos , Conformación de Ácido Nucleico , Conformación Proteica , Proteínas/química , ARN/química , Pliegue del ARNRESUMEN
Cells balance glycolysis with respiration to support their metabolic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by aerobic glycolysis or fermentation. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentation-respiration balance. The laboratory and a few other strains depended more on respiration. This trait was associated with a single nucleotide polymorphism in a conserved region of Pyk1, the sole pyruvate kinase in fission yeast. This variant reduced Pyk1 activity and glycolytic flux. Replacing the "low-activity" pyk1 allele in the laboratory strain with the "high-activity" allele was sufficient to increase fermentation and decrease respiration. This metabolic rebalancing triggered systems-level adjustments in the transcriptome and proteome and in cellular traits, including increased growth and chronological lifespan but decreased resistance to oxidative stress. Thus, low Pyk1 activity does not lead to a growth advantage but to stress tolerance. The genetic tuning of glycolytic flux may reflect an adaptive trade-off in a species lacking pyruvate kinase isoforms.
Asunto(s)
Carbono/metabolismo , Mutación Missense , Piruvato Quinasa/genética , Schizosaccharomyces/crecimiento & desarrollo , Fermentación , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glucólisis , Estrés Oxidativo , Polimorfismo de Nucleótido Simple , Proteómica , Piruvato Quinasa/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Calcineurin is an essential Ca2+-dependent phosphatase. Increased calcineurin activity is associated with α-synuclein (α-syn) toxicity, a protein implicated in Parkinson's Disease (PD) and other neurodegenerative diseases. Calcineurin can be inhibited with Tacrolimus through the recruitment and inhibition of the 12-kDa cis-trans proline isomerase FK506-binding protein (FKBP12). Whether calcineurin/FKBP12 represents a native physiologically relevant assembly that occurs in the absence of pharmacological perturbation has remained elusive. We leveraged α-syn as a model to interrogate whether FKBP12 plays a role in regulating calcineurin activity in the absence of Tacrolimus. We show that FKBP12 profoundly affects the calcineurin-dependent phosphoproteome, promoting the dephosphorylation of a subset of proteins that contributes to α-syn toxicity. Using a rat model of PD, partial elimination of the functional interaction between FKBP12 and calcineurin, with low doses of the Food and Drug Administration (FDA)-approved compound Tacrolimus, blocks calcineurin's activity toward those proteins and protects against the toxic hallmarks of α-syn pathology. Thus, FKBP12 can endogenously regulate calcineurin activity with therapeutic implications for the treatment of PD.
Asunto(s)
Calcineurina/metabolismo , Enfermedad de Parkinson/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , alfa-Sinucleína/metabolismo , Animales , Calcineurina/genética , Modelos Animales de Enfermedad , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fosfoproteínas/genética , Proteoma/genética , Ratas , Ratas Sprague-Dawley , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/genética , alfa-Sinucleína/genéticaRESUMEN
The stress sensors ATF6, IRE1, and PERK monitor deviations from homeostatic conditions in the endoplasmic reticulum (ER), a protein biogenesis compartment of eukaryotic cells. Their activation elicits unfolded protein responses (UPR) to re-establish proteostasis. UPR have been extensively investigated in cells exposed to chemicals that activate ER stress sensors by perturbing calcium, N-glycans, or redox homeostasis. Cell responses to variations in luminal load with unfolded proteins are, in contrast, poorly characterized. Here, we compared gene and protein expression profiles in HEK293 cells challenged with ER stress-inducing drugs or expressing model polypeptides. Drug titration to limit up-regulation of the endogenous ER stress reporters heat shock protein family A (Hsp70) member 5 (BiP/HSPA5) and homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERP/HERPUD1) to levels comparable with luminal accumulation of unfolded proteins substantially reduced the amplitude of both transcriptional and translational responses. However, these drug-induced changes remained pleiotropic and failed to recapitulate responses to ER load with unfolded proteins. These required unfolded protein association with BiP and induced a much smaller subset of genes participating in a chaperone complex that binds unfolded peptide chains. In conclusion, UPR resulting from ER load with unfolded proteins proceed via a well-defined and fine-tuned pathway, whereas even mild chemical stresses caused by compounds often used to stimulate UPR induce cellular responses largely unrelated to the UPR or ER-mediated protein secretion.