scholarly journals The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation

2020 ◽  
Vol 19 (11) ◽  
pp. 1896-1909
Author(s):  
Teresa M. Buck ◽  
Xuemei Zeng ◽  
Pamela S. Cantrell ◽  
Richard T. Cattley ◽  
Zikri Hasanbasri ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Proteasome System ◽  
Sterol Synthesis ◽  
High Confidence ◽  
Assembly Pathway ◽  
Affinity Isolation ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome ◽  
Erad Pathway

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin–proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques—including MS—have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

scholarly journals Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova

Reproduction ◽  
2012 ◽  
Vol 143 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Sayaka Koyanagi ◽  
Hiroko Hamasaki ◽  
Satoshi Sekiguchi ◽  
Kenshiro Hara ◽  
Yoshiyuki Ishii ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Proteomic Analysis ◽  
Ubiquitin Proteasome System ◽  
Underlying Mechanism ◽  
Wild Type ◽  
Transmission Electron ◽  
Ubiquitin Proteasome

Maternal proteins are rapidly degraded by the ubiquitin–proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficientgad. Furthermore, we assessed morphological features ingadmouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the ‘maternal antigen that embryos require’ (NLRP5 (MATER)) protein level increased significantly ingadmouse ova compared with that in wild-type mice. In an ultrastructural study,gadmouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.

scholarly journals ΔF508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression

2004 ◽  
Vol 15 (2) ◽  
pp. 563-574 ◽  
Author(s):  
Tsukasa Okiyoneda ◽  
Kazutsune Harada ◽  
Motohiro Takeya ◽  
Kaori Yamahira ◽  
Ikuo Wada ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Cystic Fibrosis Transmembrane ◽  
Erad Pathway

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ΔF508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of ΔF508 CFTR with calnexin, an ER chaperone, results in the ERAD of ΔF508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of ΔF508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ΔF508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated ΔF508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ΔF508 CFTR in the ER.

scholarly journals Neurodevelopmental Disorders (NDD) Caused by Genomic Alterations of the Ubiquitin-Proteasome System (UPS): the Possible Contribution of Immune Dysregulation to Disease Pathogenesis

2021 ◽  
Vol 14 ◽  
Author(s):  
Frédéric Ebstein ◽  
Sébastien Küry ◽  
Jonas Johannes Papendorf ◽  
Elke Krüger
Keyword(s):  
Neurodevelopmental Disorders ◽  
Ubiquitin Proteasome System ◽  
Immune Dysregulation ◽  
Innate Immune ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome ◽  
E2 Enzymes ◽  
Novel Therapeutic Strategies ◽  
The Brain

Over thirty years have passed since the first description of ubiquitin-positive structures in the brain of patients suffering from Alzheimer’s disease. Meanwhile, the intracellular accumulation of ubiquitin-modified insoluble protein aggregates has become an indisputable hallmark of neurodegeneration. However, the role of ubiquitin and a fortiori the ubiquitin-proteasome system (UPS) in the pathogenesis of neurodevelopmental disorders (NDD) is much less described. In this article, we review all reported monogenic forms of NDD caused by lesions in genes coding for any component of the UPS including ubiquitin-activating (E1), -conjugating (E2) enzymes, ubiquitin ligases (E3), ubiquitin hydrolases, and ubiquitin-like modifiers as well as proteasome subunits. Strikingly, our analysis revealed that a vast majority of these proteins have a described function in the negative regulation of the innate immune response. In this work, we hypothesize a possible involvement of autoinflammation in NDD pathogenesis. Herein, we discuss the parallels between immune dysregulation and neurodevelopment with the aim at improving our understanding the biology of NDD and providing knowledge required for the design of novel therapeutic strategies.

scholarly journals The ubiquitin proteasome system and myocardial ischemia

2013 ◽  
Vol 304 (3) ◽  
pp. H337-H349 ◽  
Author(s):  
Justine Calise ◽  
Saul R. Powell
Keyword(s):  
Myocardial Ischemia ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Future Directions ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome ◽  
The Subject ◽  
Idiopathic Cardiomyopathies ◽  
Ubiquitination Machinery

The ubiquitin proteasome system (UPS) has been the subject of intensive research over the past 20 years to define its role in normal physiology and in pathophysiology. Many of these studies have focused in on the cardiovascular system and have determined that the UPS becomes dysfunctional in several pathologies such as familial and idiopathic cardiomyopathies, atherosclerosis, and myocardial ischemia. This review presents a synopsis of the literature as it relates to the role of the UPS in myocardial ischemia. Studies have shown that the UPS is dysfunctional during myocardial ischemia, and recent studies have shed some light on possible mechanisms. Other studies have defined a role for the UPS in ischemic preconditioning which is best associated with myocardial ischemia and is thus presented here. Very recent studies have started to define roles for specific proteasome subunits and components of the ubiquitination machinery in various aspects of myocardial ischemia. Lastly, despite the evidence linking myocardial ischemia and proteasome dysfunction, there are continuing suggestions that proteasome inhibitors may be useful to mitigate ischemic injury. This review presents the rationale behind this and discusses both supportive and nonsupportive studies and presents possible future directions that may help in clarifying this controversy.

scholarly journals Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum–associated degradation

2011 ◽  
Vol 22 (16) ◽  
pp. 2797-2809 ◽  
Author(s):  
Yoshihiro Matsumura ◽  
Larry L. David ◽  
William R. Skach
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Proteasome System ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Free System ◽  
Highly Sensitive ◽  
Ubiquitin Proteasome ◽  
Cystic Fibrosis Transmembrane ◽  
Competing Pathways

The Hsp/c70 cytosolic chaperone system facilitates competing pathways of protein folding and degradation. Here we use a reconstituted cell-free system to investigate the mechanism and extent to which Hsc70 contributes to these co- and posttranslational decisions for the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR). Hsc70 binding to CFTR was destabilized by the C-terminal domain of Bag-1 (CBag), which stimulates client release by accelerating ADP-ATP exchange. Addition of CBag during CFTR translation slightly increased susceptibility of the newly synthesized protein to degradation, consistent with a profolding function for Hsc70. In contrast, posttranslational destabilization of Hsc70 binding nearly completely blocked CFTR ubiquitination, dislocation from the endoplasmic reticulum, and proteasome-mediated cleavage. This effect required molar excess of CBag relative to Hsc70 and was completely reversed by the CBag-binding subdomain of Hsc70. These results demonstrate that the profolding role of Hsc70 during cotranslational CFTR folding is counterbalanced by a dominant and essential role in posttranslational targeting to the ubiquitin-proteasome system. Moreover, the degradative outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is in turn governed by the integrated expression of regulatory cochaperones.

scholarly journals Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I

2003 ◽  
Vol 376 (3) ◽  
pp. 687-696 ◽  
Author(s):  
Claudia KITZMÜLLER ◽  
Andrea CAPRINI ◽  
Stuart E. H. MOORE ◽  
Jean-Pierre FRÉNOY ◽  
Eva SCHWAIGER ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Proteasome Inhibition ◽  
Proteasomal Degradation ◽  
Sequence Of Events ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Degradation Intermediates ◽  
Erad Pathway

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI332, which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive α1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI332 in the ERAD pathway. In a first step, the Man8 isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI332 intermediates. Some other trimmed N-glycan species, in particular Glc1Man7GlcNAc2, were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI332 accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.

The role of the ubiquitin–proteasome system in ER quality control

2005 ◽  
Vol 41 ◽  
pp. 99-112 ◽  
Author(s):  
Yihong Ye
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Mhc Class I ◽  
Heavy Chain ◽  
Ubiquitin Proteasome System ◽  
Cellular Proteins ◽  
Physiological Adaptation ◽  
Er Quality Control ◽  
Ubiquitin Proteasome

Misfolded endoplasmic reticulum (ER) proteins are eliminated by the retrotranslocation pathway in eukaryotes, which is an important physiological adaptation to ER stress. This pathway can be hijacked by certain viruses to destroy folded cellular proteins, such as MHC class I heavy chain. Recent studies have highlighted the importance of the ubiquitin–proteasome system (UPS) in this process.

scholarly journals Misfolded BiP is degraded by a proteasome-independent endoplasmic-reticulum-associated degradation pathway

2005 ◽  
Vol 387 (3) ◽  
pp. 897-903 ◽  
Author(s):  
Gerda DONOSO ◽  
Volker HERZOG ◽  
Anton SCHMITZ
Keyword(s):  
Endoplasmic Reticulum ◽  
Serine Protease ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Atp Depletion ◽  
Permeabilized Cells ◽  
Brain Microsomes ◽  
Ubiquitin Proteasome ◽  
Erad Pathway

Misfolded proteins are removed from the ER (endoplasmic reticulum) by retrotranslocation to the cytosol and degradation by the ubiquitin–proteasome system in a process designated ERAD (ER-associated degradation). Analysing the turnover of a misfolded form of the ER-resident chaperone BiP (heavy-chain binding protein) (BiPΔA), we found that the degradation of BiPΔA did not follow this general ERAD pathway. In transfected cells, BiPΔA was degraded, although proteasome-dependent ERAD was inactivated either by proteasome inhibitors or by ATP depletion. In semi-permeabilized cells, which did not support the degradation of the proteasomal substrate α1-antitrypsin, the degradation of BiPΔA was still functional, excluding the Golgi apparatus or lysosomes as the degradative compartment. The degradation of BiPΔA was recapitulated in biosynthetically loaded brain microsomes and in an extract of luminal ER proteins. In contrast with proteasome-dependent ERAD, degradation fragments were detectable inside the microsomes and in the extract, and the degradation was prevented by a serine protease inhibitor. These results show that the degradation of BiPΔA was initiated in the ER lumen by a serine protease, and support the view that proteasome-independent ERAD pathways exist.

scholarly journals Glycosylation-independent ERAD pathway serves as a backup system under ER stress

2013 ◽  
Vol 24 (20) ◽  
pp. 3155-3163 ◽  
Author(s):  
Ryo Ushioda ◽  
Jun Hoseki ◽  
Kazuhiro Nagata
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Ubiquitin Proteasome System ◽  
Stress Conditions ◽  
Backup System ◽  
Disulfide Reductase ◽  
Ubiquitin Proteasome ◽  
Erad Pathway

During endoplasmic reticulum (ER)–associated degradation (ERAD), terminally misfolded proteins are retrotranslocated from the ER to the cytosol and degraded by the ubiquitin-proteasome system. Misfolded glycoproteins are recognized by calnexin and transferred to EDEM1, followed by the ER disulfide reductase ERdj5 and the BiP complex. The mechanisms involved in ERAD of nonglycoproteins, however, are poorly understood. Here we show that nonglycoprotein substrates are captured by BiP and then transferred to ERdj5 without going through the calnexin/EDEM1 pathway; after cleavage of disulfide bonds by ERdj5, the nonglycoproteins are transferred to the ERAD scaffold protein SEL1L by the aid of BiP for dislocation into the cytosol. When glucose trimming of the N-glycan groups of the substrates is inhibited, glycoproteins are also targeted to the nonglycoprotein ERAD pathway. These results indicate that two distinct pathways for ERAD of glycoproteins and nonglycoproteins exist in mammalian cells, and these pathways are interchangeable under ER stress conditions.

scholarly journals Endoplasmic reticulum stress and the protein degradation system in ophthalmic diseases

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8638 ◽  
Author(s):  
Jing-Yao Song ◽  
Xue-Guang Wang ◽  
Zi-Yuan Zhang ◽  
Lin Che ◽  
Bin Fan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Ubiquitin Proteasome ◽  
Ophthalmic Diseases ◽  
Stress Damage ◽  
Er Homeostasis

Objective Endoplasmic reticulum (ER) stress is involved in the pathogenesis of various ophthalmic diseases, and ER stress-mediated degradation systems play an important role in maintaining ER homeostasis during ER stress. The purpose of this review is to explore the potential relationship between them and to find their equilibrium sites. Design This review illustrates the important role of reasonable regulation of the protein degradation system in ER stress-mediated ophthalmic diseases. There were 128 articles chosen for review in this study, and the keywords used for article research are ER stress, autophagy, UPS, ophthalmic disease, and ocular. Data sources The data are from Web of Science, PubMed, with no language restrictions from inception until 2019 Jul. Results The ubiquitin proteasome system (UPS) and autophagy are important degradation systems in ER stress. They can restore ER homeostasis, but if ER stress cannot be relieved in time, cell death may occur. However, they are not independent of each other, and the relationship between them is complementary. Therefore, we propose that ER stability can be achieved by adjusting the balance between them. Conclusion The degradation system of ER stress, UPS and autophagy are interrelated. Because an imbalance between the UPS and autophagy can cause cell death, regulating that balance may suppress ER stress and protect cells against pathological stress damage.

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