Widespread remodeling of proteome solubility in response to different protein homeostasis stresses

The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington’s disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.

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Widespread remodelling of proteome solubility in response to different protein homeostasis stresses

10.1101/692103 ◽

2019 ◽

Cited By ~ 1

Author(s):

Xiaojing Sui ◽

Douglas E. V. Pires ◽

Shuai Nie ◽

Giulia Vecchi ◽

Michele Vendruscolo ◽

...

Keyword(s):

Protein Complexes ◽

Protein Solubility ◽

Specific Pattern ◽

Regulatory Mechanisms ◽

Protein Homeostasis ◽

Neuro2a Cell ◽

Aggregation State ◽

Protein Deposits ◽

Almost All ◽

And Oxidative Stress

ABSTRACTThe accumulation of protein deposits in neurodegenerative diseases involves the presence of a metastable subproteome vulnerable to aggregation. To investigate this subproteome and the mechanisms that regulates it, we measured the proteome solubility of the Neuro2a cell line under protein homeostasis stresses induced by Huntington Disease proteotoxicity; Hsp70, Hsp90, proteasome and ER-mediated folding inhibition; and oxidative stress. We found one-quarter of the proteome extensively changed solubility. Remarkably, almost all the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodelling of protein complexes involved in adaptation to perturbation, most notably stress granule proteins, which responded differently to different stresses. These results indicate that the robustness of protein homeostasis relies on the absence of proteins highly vulnerable to aggregation and on large changes in aggregation state of regulatory mechanisms that restore protein solubility upon specific perturbations.

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Endoplasmic Reticulum Stress: Its Role in Disease and Novel Prospects for Therapy

Scientifica ◽

10.6064/2012/857516 ◽

2012 ◽

Vol 2012 ◽

pp. 1-26 ◽

Cited By ~ 113

Author(s):

Axel H. Schönthal

Keyword(s):

Endoplasmic Reticulum ◽

Protein Folding ◽

Er Stress ◽

Prescription Drugs ◽

Pancreatic Beta Cells ◽

Cell Function ◽

Tumor Cell Death ◽

Cellular Mechanisms ◽

Pharmacological Agents ◽

The Unfolded Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle required for lipid biosynthesis, calcium storage, and protein folding and processing. A number of physiological and pathological conditions, as well as a variety of pharmacological agents, are able to disturb proper ER function and thereby cause ER stress, which severely impairs protein folding and therefore poses the risk of proteotoxicity. Specific triggers for ER stress include, for example, particular intracellular alterations (e.g., calcium or redox imbalances), certain microenvironmental conditions (e.g., hypoglycemia, hypoxia, and acidosis), high-fat and high-sugar diet, a variety of natural compounds (e.g., thapsigargin, tunicamycin, and geldanamycin), and several prescription drugs (e.g., bortezomib/Velcade, celecoxib/Celebrex, and nelfinavir/Viracept). The cell reacts to ER stress by initiating a defensive process, called the unfolded protein response (UPR), which is comprised of cellular mechanisms aimed at adaptation and safeguarding cellular survival or, in cases of excessively severe stress, at initiation of apoptosis and elimination of the faulty cell. In recent years, this dichotomic stress response system has been linked to several human diseases, and efforts are underway to develop approaches to exploit ER stress mechanisms for therapy. For example, obesity and type 2 diabetes have been linked to ER stress-induced failure of insulin-producing pancreatic beta cells, and current research efforts are aimed at developing drugs that ameliorate cellular stress and thereby protect beta cell function. Other studies seek to pharmacologically aggravate chronic ER stress in cancer cells in order to enhance apoptosis and achieve tumor cell death. In the following, these principles will be presented and discussed.

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Pulse labeling reveals the tail end of protein folding by proteome profiling

10.1101/2021.03.28.437442 ◽

2021 ◽

Author(s):

Mang Zhu ◽

Erich R. Kuechler ◽

Nikolay Stoynov ◽

Joerg Gsponer ◽

Thibault Mayor

Keyword(s):

Mass Spectrometry ◽

Protein Folding ◽

Heat Stress ◽

Protein Complexes ◽

Protein Sequences ◽

Protein Homeostasis ◽

Native State ◽

Binding Motifs ◽

Specific Subset ◽

Β Sheet

SummaryAccurate and efficient folding of nascent protein sequences into their native state requires support from the protein homeostasis network. Herein we probed which newly translated proteins are less thermostable to infer which polypeptides require more time to fold within the proteome. Specifically, we determined which of these proteins were more susceptible to misfolding and aggregation under heat stress using pulse SILAC coupled mass spectrometry. These proteins are abundant, short, and highly structured. Notably these proteins display a tendency to form β-sheet structures, a configuration which typically requires more time for folding, and were enriched for Hsp70/Ssb and TRiC/CCT binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides were also more often components of stable protein complexes in comparison to other proteins. All evidence combined suggests that a specific subset of newly translated proteins requires more time following synthesis to reach a thermostable native state in the cell.

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NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

The Journal of Cell Biology ◽

10.1083/jcb.201006121 ◽

2010 ◽

Vol 191 (6) ◽

pp. 1113-1125 ◽

Cited By ~ 187

Author(s):

Gang Li ◽

Christopher Scull ◽

Lale Ozcan ◽

Ira Tabas

Keyword(s):

Oxidative Stress ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Cellular Mechanisms ◽

Apoptosis Pathway ◽

Stress Oxidative ◽

Induced Apoptosis ◽

And Oxidative Stress

Endoplasmic reticulum (ER)–induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress–apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA–dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.

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Antimyeloma In Vitro Activity Of 15-Deoxy-D12,14-Prostaglandin J2 Is Associated With Endoplasmic Reticulum Stress In a Reactive Oxygen Species-Dependent Fashion

Blood ◽

10.1182/blood.v122.21.4913.4913 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4913-4913

Author(s):

Ana Paula Demasi ◽

Marcelo Henrique Napimoga ◽

Elizabeth Ferreira Martinez ◽

Adriana Silva Santos Duarte ◽

Fernando V Pericole ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Multiple Myeloma ◽

Endoplasmic Reticulum ◽

Protein Folding ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Research Funding ◽

Misfolded Proteins ◽

Oxygen Species

Abstract Multiple myeloma (MM) is an incurable malignancy characterized by the accumulation of terminally differentiated plasma cells in the bone marrow, usually accompanied by continuous monoclonal immunoglobulin production. This production requires proper folding and assembly of immunoglobulin chains, conducted by disulfide bonds formation in the endoplasmic reticulum (ER). Disulfide bonds are formed via electron transfer from thiol groups in a protein relay system, from protein disulfide isomerase (PDI) to ER oxidoreductase1 (ERO1) and finally to molecular oxygen, generating one molecule of hydrogen peroxide (H2O2) as secondary product for each disulfide bond formed. The high ER protein-folding demand usually placed on MM cells predisposes them to accumulation of unfolded proteins, which results in ER stress and also oxidative stress, both capable to induce cell death. Cellular defense systems for these specific forms of stress, known as the unfolded protein response (UPR) and oxidative stress response, have been explored as therapeutic targets for MM. 15-Deoxy-D12,14-prostaglandin J2 (15d-PGJ2) is a member of J2 series of PGs, characterized by the presence of an electrophilic carbonyl group in the cyclopentenone ring, which confers them biological properties that are different from those of other components of the PG family. These include anti-inflammatory, anti-proliferative and pro-apoptotic activities, depending on the concentration and cell type. Starting from the observation that15d-PGJ2 is emerging as the most potent antineoplastic agent of the J2 series of PGs, in the present study we investigated its effect on MM cells. We found that 15d-PGJ2 displayed superior cytotoxicity than dexamethasone in MM cell lines. Using ﬂow cytometry, we demonstrated intracellular reactive oxygen species (ROS) formation in these cells after 15d-PGJ2 treatment, which was associated with an augmented ratio of oxidized to reduced glutathione (GSH), indicating that the cells were under oxidative stress. Due to its electrophilic property, it has been suggested that 15d-PGJ2 may directly react with GSH and/or other thiol compounds. Such reactions would deplete essential cellular antioxidants thereby leading to oxidative stress in MM cells. Results from qPCR analysis showed that 15d-PGJ2 induces transcription of UPR genes, including GRP78, GRP94, EDEM1 and also PRDX4. GRP78/BiP and GRP94 are ER-resident chaperones, members of the heat shock protein (HSP) 70 family and HSP 90 family, respectively. These chaperones were described to bind to immunoglobulin chains assisting their assembly. The ER degradation-enhancer mannosidase-like protein 1 (Edem1) is involved in the degradation of misfolded proteins to prevent their aggregation and cytotoxicity. PRDX4 encodes the ER-localized peroxiredoxin (Prx) IV, an enzyme not yet recognized as an UPR component, but recently demonstrated to use the H2O2 produced by ERO1 during disulfide bond formation to generate another pair of disulfide, with the additional benefit of reducing H2O2 to water. Increased transcription of these genes indicates an attempt to preserve ER-processing function and MM cells survival in response to accumulated misfolded proteins. In this regard, 15d-PGJ2-induced oxidative stress, by provoking oxidative modification of proteins, could have intensified the formation of misfolded proteins, thereby activating UPR signaling. In agreement with 15d-PGJ2-mediated accumulation of misfolded proteins, which are prevented from progressing further along the secretory pathway, our results from ELISA have shown a massive decrease in the concentration of light chain immunoglobulin secreted by MM cells in the presence of 15d-PGJ2, but not dexamethasone. These data suggest that 15d-PGJ2 favors ROS accumulation and protein misfolding, especially threatening the survival of cells that have a high protein-folding load and/or are susceptible to oxidative stress. In this sense, multiple myeloma and related disorders may benefit from 15d-PGJ2 therapy. Disclosures: Demasi: FAPESP: Research Funding. Napimoga:FAPESP: Research Funding.

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Endoplasmic Reticulum Stress and Unfolded Protein Response Signaling in Plants

International Journal of Molecular Sciences ◽

10.3390/ijms23020828 ◽

2022 ◽

Vol 23 (2) ◽

pp. 828

Author(s):

Hakim Manghwar ◽

Jianming Li

Keyword(s):

Endoplasmic Reticulum ◽

Protein Folding ◽

Er Stress ◽

Unfolded Protein Response ◽

Defense Mechanisms ◽

Protein Complexes ◽

Unfolded Protein ◽

Subcellular Organelle ◽

The Unfolded Protein Response ◽

Protein Response

Plants are sensitive to a variety of stresses that cause various diseases throughout their life cycle. However, they have the ability to cope with these stresses using different defense mechanisms. The endoplasmic reticulum (ER) is an important subcellular organelle, primarily recognized as a checkpoint for protein folding. It plays an essential role in ensuring the proper folding and maturation of newly secreted and transmembrane proteins. Different processes are activated when around one-third of newly synthesized proteins enter the ER in the eukaryote cells, such as glycosylation, folding, and/or the assembling of these proteins into protein complexes. However, protein folding in the ER is an error-prone process whereby various stresses easily interfere, leading to the accumulation of unfolded/misfolded proteins and causing ER stress. The unfolded protein response (UPR) is a process that involves sensing ER stress. Many strategies have been developed to reduce ER stress, such as UPR, ER-associated degradation (ERAD), and autophagy. Here, we discuss the ER, ER stress, UPR signaling and various strategies for reducing ER stress in plants. In addition, the UPR signaling in plant development and different stresses have been discussed.

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Ero1–PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2011.0403 ◽

2013 ◽

Vol 368 (1617) ◽

pp. 20110403 ◽

Cited By ~ 45

Author(s):

Adam M. Benham ◽

Marcel van Lith ◽

Roberto Sitia ◽

Ineke Braakman

Keyword(s):

Endoplasmic Reticulum ◽

Protein Folding ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Secretory Pathway ◽

Protein Complexes ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Terminal Electron Acceptor ◽

Post Translational Modifications

The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide bonds. These are introduced into client proteins by ER resident oxidoreductases, including ER oxidoreductin 1 (Ero1). Ero1 is usually considered to function in a linear pathway, by ‘donating’ a disulfide bond to protein disulfide isomerase (PDI) and receiving electrons that are passed on to the terminal electron acceptor molecular oxygen. PDI engages with a range of clients as the direct catalyst of disulfide bond formation, isomerization or reduction. In this paper, we will consider the interactions of Ero1 with PDI family proteins and chaperones, highlighting the effect that redox flux has on Ero1 partnerships. In addition, we will discuss whether higher order protein complexes play a role in Ero1 function.

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Extending the proteomic characterization of Candida albicans exposed to stress and apoptotic inducers through data-independent acquisition mass espectrometry

Access Microbiology ◽

10.1099/acmi.cc2021.po0087 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Ahinara Amador-García ◽

Johan Malmström ◽

Lucia Monteoliva ◽

Concha Gil

Keyword(s):

Oxidative Stress ◽

Protein Folding ◽

Candida Albicans ◽

Stress Response ◽

Oxidative Stress Response ◽

Antifungal Drugs ◽

Proteasome Activity ◽

Main Role ◽

Data Independent Acquisition ◽

Almost All

Candida albicans is a commensal fungus that causes systemic infections in immunosuppressed patients. In order to deal with the changing environment during commensalism or infection, C. albicans must reprogram its proteome. Characterizing the stress-induced changes in the proteome that C. albicans uses to survive should be very useful in the development of new antifungal drugs. We studied the C. albicans global proteome after exposure to hydrogen peroxide (H2O2) and acetic acid (AA), using a DIA-MS strategy. More than 2000 C. albicans proteins were quantified using an ion library previously constructed using DDA-MS. C. albicans responded to treatment with H2O2 with an increase in the abundance of many proteins involved in the oxidative stress response, protein folding and proteasome-dependent catabolism, which led to an increased proteasome activity. The data revealed a previously unknown key role for Prn1, a protein similar to pirins, in the oxidative stress response. Treatment with AA resulted in a general decrease in the abundance of proteins involved in amino acid biosynthesis, protein folding, and rRNA processing. Almost all proteasome proteins declined, as did proteasome activity. Apoptosis was observed after treatment with H2O2, but not AA. A targeted proteomic study of 32 proteins related to apoptosis in yeast supported the results found by DIA-MS and allowed the creation of an efficient method to quantify relevant proteins after treatment with stressors (H2O2, AA, and amphotericin B). This approach also uncovered a main role for Oye32, an oxidoreductase, suggesting this protein as a possible apoptotic marker common to many stressors.

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Engineering ER-stress dependent non-conventional mRNA splicing

eLife ◽

10.7554/elife.35388 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 10

Author(s):

Weihan Li ◽

Voytek Okreglak ◽

Jirka Peschek ◽

Philipp Kimmig ◽

Meghan Zubradt ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Protein Folding ◽

Er Stress ◽

Schizosaccharomyces Pombe ◽

Transcriptional Response ◽

Structural Features ◽

Mrna Splicing ◽

Protein Homeostasis ◽

Stress Dependent

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un- or misfolded proteins. The ER membrane-resident kinase/RNase Ire1 maintains ER protein homeostasis through two fundamentally distinct processes. First, Ire1 can initiate a transcriptional response through a non-conventional mRNA splicing reaction to increase the ER folding capacity. Second, Ire1 can decrease the ER folding burden through selective mRNA decay. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two Ire1 functions have been evolutionarily separated. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we engineered an S. pombe Ire1 cleavage substrate into a splicing substrate, which confers S. pombe with both Ire1 functional outputs.

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