ER stress causes widespread protein aggregation and prion formation

Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.

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How to design an optimal sensor network for the unfolded protein response

Molecular Biology of the Cell ◽

10.1091/mbc.e18-01-0060 ◽

2018 ◽

Vol 29 (25) ◽

pp. 3052-3062 ◽

Cited By ~ 3

Author(s):

Wylie Stroberg ◽

Hadar Aktin ◽

Yonatan Savir ◽

Santiago Schnell

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

Cellular Protein ◽

Protein Accumulation ◽

Protein Homeostasis ◽

Unfolded Protein ◽

Stress Sensing ◽

Coarse Model ◽

The Unfolded Protein Response ◽

Protein Response

Cellular protein homeostasis requires continuous monitoring of stress in the endoplasmic reticulum (ER). Stress-detection networks control protein homeostasis by mitigating the deleterious effects of protein accumulation, such as aggregation and misfolding, with precise modulation of chaperone production. Here, we develop a coarse model of the unfolded protein response in yeast and use multi-objective optimization to determine which sensing and activation strategies optimally balance the trade-off between unfolded protein accumulation and chaperone production. By comparing a stress-sensing mechanism that responds directly to the level of unfolded protein in the ER to a mechanism that is negatively regulated by unbound chaperones, we show that chaperone-mediated sensors are more efficient than sensors that detect unfolded proteins directly. This results from the chaperone-mediated sensor having separate thresholds for activation and deactivation. Finally, we demonstrate that a sensor responsive to both unfolded protein and unbound chaperone does not further optimize homeostatic control. Our results suggest a strategy for designing stress sensors and may explain why BiP-mitigated ER stress-sensing networks have evolved.

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How to design an optimal sensor network for the unfolded protein response

10.1101/396614 ◽

2018 ◽

Author(s):

Wylie Stroberg ◽

Hadar Aktin ◽

Yonatan Savir ◽

Santiago Schnell

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

Cellular Protein ◽

Protein Accumulation ◽

Protein Homeostasis ◽

Unfolded Protein ◽

Stress Sensing ◽

Coarse Model ◽

The Unfolded Protein Response ◽

Protein Response

AbstractCellular protein homeostasis requires continuous monitoring of stress in the endoplasmic reticulum (ER). Stress detection networks control protein homeostasis by mitigating the deleterious effects of protein accumulation, such as aggregation and misfolding, with precise modulation of chaperone production. Here, we develop a coarse model of the unfolded protein response in yeast, and use multi-objective optimization to determine which sensing and activation strategies optimally balance the trade-off between unfolded protein accumulation and chaperone production. By comparing a stress-sensing mechanism that responds directly to the level of unfolded protein in the ER to a mechanism that is negatively regulated by unbound chaperones, we show that chaperone-mediated sensors are more efficient than sensors that detect unfolded proteins directly. This results from the chaperone-mediated sensor having separate thresholds for activation and deactivation. Lastly, we demonstrate that a sensor responsive to both unfolded protein and unbound chaperone does not further optimize homeostatic control. Our results suggest a strategy for designing stress sensors and may explain why BiP-mitigated ER stress sensing networks have evolved.

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Two Distinctly Localized P-Type ATPases Collaborate to Maintain Organelle Homeostasis Required for Glycoprotein Processing and Quality Control

Molecular Biology of the Cell ◽

10.1091/mbc.02-06-0090 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3955-3966 ◽

Cited By ~ 61

Author(s):

Shilpa Vashist ◽

Christian G. Frank ◽

Claude A. Jakob ◽

Davis T.W. Ng

Keyword(s):

Quality Control ◽

Unfolded Protein Response ◽

Secretory Pathway ◽

Ion Homeostasis ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Coa Reductase ◽

Protein Response ◽

P Type ◽

Er Homeostasis

Membrane transporter proteins are essential for the maintenance of cellular ion homeostasis. In the secretory pathway, the P-type ATPase family of transporters is found in every compartment and the plasma membrane. Here, we report the identification of COD1/SPF1(control of HMG-CoA reductase degradation/SPF1) through genetic strategies intended to uncover genes involved in protein maturation and endoplasmic reticulum (ER)-associated degradation (ERAD), a quality control pathway that rids misfolded proteins. Cod1p is a putative ER P-type ATPase whose expression is regulated by the unfolded protein response, a stress-inducible pathway used to monitor and maintain ER homeostasis. COD1 mutants activate the unfolded protein response and are defective in a variety of functions apart from ERAD, which further support a homeostatic role.COD1 mutants display phenotypes similar to strains lacking Pmr1p, a Ca2+/Mn2+pump that resides in the medial-Golgi. Because of its localization, the previously reported role of PMR1 in ERAD was somewhat enigmatic. A clue to their respective roles came from observations that the two genes are not generally required for ERAD. We show that the specificity is rooted in a requirement for both genes in protein-linked oligosaccharide trimming, a requisite ER modification in the degradation of some misfolded glycoproteins. Furthermore, Cod1p, like Pmr1p, is also needed for the outer chain modification of carbohydrates in the Golgi apparatus despite its ER localization. In strains deleted of both genes, these activities are nearly abolished. The presence of either protein alone, however, can support partial function for both compartments. Taken together, our results reveal an interdependent relationship between two P-type ATPases to maintain homeostasis of the organelles where they reside.

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70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab70 ◽

2017 ◽

Vol 29 (1) ◽

pp. 142

Author(s):

K. Gutierrez ◽

W. G. Glanzner ◽

N. Dicks ◽

R. C. Bohrer ◽

L. G. Currin ◽

...

Keyword(s):

Er Stress ◽

Gene Editing ◽

Early Embryo ◽

Early Embryo Development ◽

Unfolded Protein ◽

Guide Rnas ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9) + 1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9 + 1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9 + sgRNA-1 + sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1 + sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Student’s t-test or Tukey–Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a = 59.8 ± 4.9%, b = 58.8 ± 5.3%, c = 68.86 ± 2.2%, d = 66.4 ± 5.9%, and e = 70.10 ± 1.9%), but development to the blastocyst stage was substantially reduced (P < 0.05) in the groups injected with Cas9 + sgRNAs (a = 18 ± 4.5%, b = 16 ± 1.5%, and c = 5.3 ± 2.8%) compared with controls (d = 33.7 ± 6.2% and e = 31.4 ± 1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9 + sgRNA-1 + sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.

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The Impact of Endoplasmic Reticulum-Associated Protein Modifications, Folding and Degradation on Lung Structure and Function

Frontiers in Physiology ◽

10.3389/fphys.2021.665622 ◽

2021 ◽

Vol 12 ◽

Author(s):

Emily M. Nakada ◽

Rui Sun ◽

Utako Fujii ◽

James G. Martin

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Unfolded Protein ◽

Lung Structure ◽

Selective Translation ◽

The Unfolded Protein Response ◽

And Function ◽

Protein Response ◽

Er Homeostasis ◽

The Impact

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and induces the unfolded protein response (UPR) and other mechanisms to restore ER homeostasis, including translational shutdown, increased targeting of mRNAs for degradation by the IRE1-dependent decay pathway, selective translation of proteins that contribute to the protein folding capacity of the ER, and activation of the ER-associated degradation machinery. When ER stress is excessive or prolonged and these mechanisms fail to restore proteostasis, the UPR triggers the cell to undergo apoptosis. This review also examines the overlooked role of post-translational modifications and their roles in protein processing and effects on ER stress and the UPR. Finally, these effects are examined in the context of lung structure, function, and disease.

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Endoplasmic reticulum stress and the development of endothelial dysfunction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00437.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. H355-H367 ◽

Cited By ~ 34

Author(s):

M. L. Battson ◽

D. M. Lee ◽

C. L. Gentile

Keyword(s):

Endoplasmic Reticulum ◽

Endothelial Dysfunction ◽

Er Stress ◽

Critical Role ◽

Unfolded Protein ◽

Vasoactive Substances ◽

Important Regulator ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

The vascular endothelium plays a critical role in cardiovascular homeostasis, and thus identifying the underlying causes of endothelial dysfunction has important clinical implications. In this regard, the endoplasmic reticulum (ER) has recently emerged as an important regulator of metabolic processes. Dysfunction within the ER, broadly termed ER stress, evokes the unfolded protein response (UPR), an adaptive pathway that aims to restore ER homeostasis. Although the UPR is the first line of defense against ER stress, chronic activation of the UPR leads to cell dysfunction and death and has recently been implicated in the pathogenesis of endothelial dysfunction. Numerous risk factors for endothelial dysfunction can induce ER stress, which may in turn disrupt endothelial function via direct effects on endothelium-derived vasoactive substances or by activating other pathogenic cellular networks such as inflammation and oxidative stress. This review summarizes the available data linking ER stress to endothelial dysfunction.

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Different Forms of ER Stress in Chondrocytes Result in Short Stature Disorders and Degenerative Cartilage Diseases: New Insights by Cartilage-Specific ERp57 Knockout Mice

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/8421394 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 5

Author(s):

Yvonne Rellmann ◽

Rita Dreier

Keyword(s):

Er Stress ◽

Short Stature ◽

Knockout Mice ◽

Skeletal Development ◽

Unfolded Protein ◽

Cartilage Diseases ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis ◽

Protein Disulfide

Cartilage is essential for skeletal development by endochondral ossification. The only cell type within the tissue, the chondrocyte, is responsible for the production of macromolecules for the extracellular matrix (ECM). Before proteins and proteoglycans are secreted, they undergo posttranslational modification and folding in the endoplasmic reticulum (ER). However, the ER folding capacity in the chondrocytes has to be balanced with physiological parameters like energy and oxygen levels. Specific cellular conditions, e.g., a high protein demand, or pathologic situations disrupt ER homeostasis and lead to the accumulation of poorly folded or misfolded proteins. This state is called ER stress and induces a cellular quality control system, the unfolded protein response (UPR), to restore homeostasis. Different mouse models with ER stress in chondrocytes display comparable skeletal phenotypes representing chondrodysplasias. Therefore, ER stress itself seems to be involved in the pathogenesis of these diseases. It is remarkable that chondrodysplasias with a comparable phenotype arise independent from the sources of ER stress, which are as follows: (1) mutations in ECM proteins leading to aggregation, (2) deficiencies in ER chaperones, (3) mutations in UPR signaling factors, or (4) deficiencies in the degradation of aggregated proteins. In any case, the resulting UPR substantially impairs ECM protein synthesis, chondrocyte proliferation, and/or differentiation or regulation of autophagy and apoptosis. Notably, chondrodysplasias arise no matter if single or multiple events are affected. We analyzed cartilage-specific ERp57 knockout mice and demonstrated that the deficiency of this single protein disulfide isomerase, which is responsible for formation of disulfide bridges in ECM glycoproteins, is sufficient to induce ER stress and to cause an ER stress-related bone phenotype. These mice therefore qualify as a novel model for the analysis of ER stress in chondrocytes. They give new insights in ER stress-related short stature disorders and enable the analysis of ER stress in other cartilage diseases, such as osteoarthritis.

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Coordination of stress, Ca2+, and immunogenic signaling pathways by PERK at the endoplasmic reticulum

Biological Chemistry ◽

10.1515/hsz-2016-0108 ◽

2016 ◽

Vol 397 (7) ◽

pp. 649-656 ◽

Cited By ~ 10

Author(s):

Alexander R. van Vliet ◽

Abhishek D. Garg ◽

Patrizia Agostinis

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Signaling Pathways ◽

Unfolded Protein ◽

Independent Functions ◽

Intracellular Ca ◽

Stress Signals ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

AbstractThe endoplasmic reticulum (ER) is the main coordinator of intracellular Ca2+signaling, protein synthesis, and folding. The ER is also implicated in the formation of contact sites with other organelles and structures, including mitochondria, plasma membrane (PM), and endosomes, thereby orchestrating through interorganelle signaling pathways, a variety of cellular responses including Ca2+homeostasis, metabolism, and cell death signaling. Upon loss of its folding capacity, incited by a number of stress signals including those elicited by various anticancer therapies, the unfolded protein response (UPR) is launched to restore ER homeostasis. The ER stress sensor protein kinase RNA-like ER kinase (PERK) is a key mediator of the UPR and its role during ER stress has been largely recognized. However, growing evidence suggests that PERK may govern signaling pathways through UPR-independent functions. Here, we discuss emerging noncanonical roles of PERK with particular relevance for the induction of danger or immunogenic signaling and interorganelle communication.

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The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

10.26686/wgtn.17008045 ◽

2021 ◽

Author(s):

◽

Peter William Bircham

Keyword(s):

Er Stress ◽

High Throughput ◽

Secretory Pathway ◽

Misfolded Proteins ◽

Phenotypic Changes ◽

Unfolded Protein ◽

Gfp Reporter ◽

Reporter Systems ◽

The Unfolded Protein Response ◽

Protein Response

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>

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The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins

10.1101/2021.05.19.444788 ◽

2021 ◽

Author(s):

Katharina Knoeringer ◽

Carina Groh ◽

Lena Kraemer ◽

Kevin C Stein ◽

Katja G Hansen ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Unfolded Protein Response ◽

Mitochondrial Membrane ◽

Cellular Protein ◽

Mitochondrial Proteins ◽

Protein Homeostasis ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPR-ER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The levels of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPR-ER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cannot be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

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