An Hsp90 co-chaperone links protein folding and degradation and is part of a conserved protein quality control
Frederik EiseleAnna Maria Eisele-BürgerXinxin HaoLisa Larsson BerglundJohanna L. HöögBeidong LiuThomas Nyström
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In this paper, we show that the essential Hsp90 co-chaperone Sgt1 is a member of a general protein quality control network that links folding and degradation through its participation in the degradation of misfolded proteins both in the cytosol and the endoplasmic reticulum (ER). Sgt1-dependent protein degradation acts in a parallel pathway to the ubiquitin ligase (E3) and ubiquitin chain elongase (E4), Hul5, and overproduction of Hul5 partly suppresses defects in cells with reduced Sgt1 activity. Upon proteostatic stress, Sgt1 accumulates transiently, in an Hsp90- and proteasome-dependent manner, with quality control sites (Q-bodies) of both yeast and human cells that co-localize with Vps13, a protein that creates organelle contact sites. Misfolding disease proteins, such as synphilin-1 involved in Parkinson's disease, are also sequestered to these compartments and require Sgt1 for their clearance.Keywords:
Chaperone (clinical)
Aggresome
Endoplasmic-reticulum-associated protein degradation
Protein Degradation
Ubiquitin-Protein Ligases
Organelle
Protein quality
Proteostasis
Co-chaperone
Endoplasmic-reticulum-associated protein degradation
Proteostasis
Aggresome
Protein Degradation
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Protein quality control and subsequent elimination of terminally misfolded proteins occurs via the ubiquitin–proteasome system. Tagging of misfolded proteins with ubiquitin for degradation depends on a cascade of reactions involving an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). While ubiquitin ligases responsible for targeting misfolded secretory proteins to proteasomal degradation (ERAD) have been uncovered, no such E3 enzymes have been found for elimination of misfolded cytoplasmic proteins in yeast. Here we report on the discovery of Ubr1, the E3 ligase of the N‐end rule pathway, to be responsible for targeting misfolded cytosoplasmic protein to proteasomal degradation.
Endoplasmic-reticulum-associated protein degradation
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Ubiquitin-Protein Ligases
Ubiquitin-conjugating enzyme
DDB1
Protein Degradation
F-box protein
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Protein misfolding is a major driver of ageing‐associated frailty and disease pathology. Although all cells possess multiple, well‐characterised protein quality control systems to mitigate the toxicity of misfolded proteins, how they are integrated to maintain protein homeostasis (‘proteostasis’) in health—and how their disintegration contributes to disease—is still an exciting and fast‐paced area of research. Under physiological conditions, the predominant route for misfolded protein clearance involves ubiquitylation and proteasome‐mediated degradation. When the capacity of this route is overwhelmed—as happens during conditions of acute environmental stress, or chronic ageing‐related decline—alternative routes for protein quality control are activated. In this review, we summarise our current understanding of how proteasome‐targeted misfolded proteins are retrafficked to alternative protein quality control routes such as juxta‐nuclear sequestration and selective autophagy when the ubiquitin–proteasome system is compromised. We also discuss the molecular determinants of these alternative protein quality control systems, attempt to clarify distinctions between various cytoplasmic spatial quality control inclusion bodies (e.g., Q‐bodies, p62 bodies, JUNQ, aggresomes, and aggresome‐like induced structures ‘ALIS’), and speculate on emerging concepts in the field that we hope will spur future research—with the potential to benefit the rational development of healthy ageing strategies.
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Proteostasis
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Significance Ubiquitin (Ub) conjugation triggers protein degradation by the proteasome. Here we describe an unexplored feature of the Ub conjugation system that entails differential activation of an E2-conjugating enzyme by its cognate E3 Ub ligases. In vitro and in vivo analyses of activity-reducing mutants of the yeast Ub-conjugating (Ubc) enzyme, Ubc7, demonstrated selective activation by one of its two cognate E3 ligases, Hrd1, but not by the other, Doa10. Supported by structural modeling of the RING:Ubc7∼Ub complex, these findings are consistent with a model whereby the E2∼Ub transition state depends on noncovalent interactions between helix α2 of Ubc7 and Ub that are differentially stabilized by the two E3 RING domains. This differential activation represents a previously unidentified mechanism for regulating protein ubiquitylation.
Endoplasmic-reticulum-associated protein degradation
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Misfolded/mutated proteins are identified by the endoplasmic reticulum (ER) quality control system (ER-QC) and eliminated through the ER-associated protein degradation (ERAD) pathway. The presence of structural defects identifies these proteins as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. ER-associated E3 ubiquitin ligases are key components of the ERAD machinery, and distinct E3 ligases seem to control specific ERAD pathways.
In human muscles, mutations on sarcoglycans, proteins that form a tetramer complex (alpha-beta-gamma-delta) associated to dystrophin, lead to a Limb Girdle Muscolar Dystrophy (LGMD). It has been demonstrated that the V247M alpha-sarcoglycan mutant, a type I membrane protein with a luminal defect, is ubiquitinated and degraded by the proteasome, the last ERAD component. Aim of this project is to investigate ERAD components involved in recognition, ubiquitination and retrotraslocation of the mutated protein. The attention has been focused particullary on the E3 ligases that seem to be crucial factors assuring both selectivity and specificity to ERAD pathways. Emerging literature regarding several diseases involving mutated/misfolded proteins augurs well for treatments that involved common or peculiar ER-QC and ERAD steps. Due to the fact that until now, there is no known therapy for muscular dystrophies, I believe this study is relevant both to disclose an important biological process but also to identify possible molecular targets to treat sarcoglycanopathies.
A set of ER chaperones and lectins recognize and transport misfolded proteins to the site of dislocation. Among these, I have demonstrated that the chaperone GRP94 is probably involved in V247M mutant recognition, while BiP and OS9 do not seem to be implicated, however, their role is still under investigation. Usually gp78 and HRD1 are the E3 ligases involved in the ERAD-L pathway of model substrates. By using mutated variants of, or siRNAs for these E3 candidates, my results show that HRD1, but not gp78, is specifically involved in the disposal of the alpha-sarcoglycan mutant. In addition, the E2 ubiquitin-conjugase enzyme UBC6e and the cargo receptor SEL1L, well-known HRD1 partners, cooperate in the disposal of V247M alpha-SG. Immunoprecipitation experiments validated the interaction of HRD1, UBC6e and SEL1L with the alpha-SG mutant. Moreover another E3 ligase, RFP2, is involved in the degradation of V247M alpha-SG being able both to co-immunoprecipitate with the protein and to block its disposal, if a mutated variant is expressed. The driving force to eradicate misfolded membrane proteins from the ER is usually provided by the AAA-ATPase p97. My experiments demonstrated that p97, together with the ER-associated Derlin-1, form the so-called dislocon, the proteinaceous environment for the hydrophilic luminal domain of V247M mutant to cross the ER membrane.
These results describe for the first time the ERAD pathway for the disposal of the type I membrane protein V247M alpha-sarcoglycan, a pathway leaded by the E3 ligase HRD1. Moerover, recognition and delivery to proteasome of this mutant is also assisted by the E3 ubiquitin ligase RFP2.
In collaboration with Dr. R. Sacchetto (Dept. of Veterinary), I also carried out a study aimed to check whether the R164H mutant of the polytopic membrane protein SERCA1a is also an ERAD client. Mutations in SERCA1a are responsible for Brody Disease, a human inherited congenital disorder that affects skeletal muscles, because of SERCA1a loss of function. A similar muscular disorder, named congenital Pseudomyotonia, has been described in the Italian Chianina cattle. In affected animals and Brody’s patients, decreased calcium ATPase activity perfectly correlates with reduced expression of SERCA1a protein. I demonstrate that inhibition of proteasome not only rescued the expression of R164H SERCA1a mutant transfected in HEK-293 cells, but also restored the enzymatic activity.
Endoplasmic-reticulum-associated protein degradation
Ubiquitin-Protein Ligases
Protein Degradation
Limb-girdle muscular dystrophy
Chaperone (clinical)
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Endoplasmic-reticulum-associated protein degradation
Ubiquitin-conjugating enzyme
Ubiquitin-Protein Ligases
Protein Degradation
F-box protein
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Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases. Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. Protein folding is governed solely by the protein itself, scientists discovered that some proteins have helped in the process called chaperones. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Misfolding of newly formed proteins not only results in a loss of physiological function of the protein but also may lead to the intra- or extra- cellular accumulation of that protein. A number of diseases have been shown to be characterised by the accumulation of misfolded proteins, notable example being Alzheimer's disease.
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Protein quality
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Physiological adaptation to proteotoxic stress in the endoplasmic reticulum (ER) requires retrotranslocation of misfolded proteins into the cytoplasm for ubiquitination and elimination by ER-associated degradation (ERAD). A surprising paradox emerging from recent studies is that ubiquitin ligases (E3s) and deubiquitinases (DUBs), enzymes with opposing activities, can both promote ERAD. Here we demonstrate that the ERAD E3 gp78 can ubiquitinate not only ERAD substrates, but also the machinery protein Ubl4A, a key component of the Bag6 chaperone complex. Remarkably, instead of targeting Ubl4A for degradation, polyubiquitination is associated with irreversible proteolytic processing and inactivation of Bag6. Importantly, we identify USP13 as a gp78-associated DUB that eliminates ubiquitin conjugates from Ubl4A to maintain the functionality of Bag6. Our study reveals an unexpected paradigm in which a DUB prevents undesired ubiquitination to sharpen substrate specificity for an associated ubiquitin ligase partner and to promote ER quality control.
Endoplasmic-reticulum-associated protein degradation
Ubiquitin-Protein Ligases
Chaperone (clinical)
Protein Degradation
AAA proteins
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Citations (71)
Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases. Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. Protein folding is governed solely by the protein itself, scientists discovered that some proteins have helped in the process called chaperones. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Misfolding of newly formed proteins not only results in a loss of physiological function of the protein but also may lead to the intra- or extra- cellular accumulation of that protein. A number of diseases have been shown to be characterised by the accumulation of misfolded proteins, notable example being Alzheimer's disease.
Aggresome
Co-chaperone
Chaperone (clinical)
Protein quality
Folding (DSP implementation)
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Aberrant endoplasmic reticulum (ER) proteins are eliminated by ER-associated degradation (ERAD). This process involves protein retrotranslocation into the cytosol, ubiquitylation, and proteasomal degradation. ERAD substrates are classified into three categories based on the location of their degradation signal/degron: ERAD-L (lumen), ERAD-M (membrane), and ERAD-C (cytosol) substrates. In Saccharomyces cerevisiae, the membrane proteins Hrd1 and Doa10 are the predominant ERAD ubiquitin-protein ligases (E3s). The current notion is that ERAD-L and ERAD-M substrates are exclusively handled by Hrd1, whereas ERAD-C substrates are recognized by Doa10. In this paper, we identify the transmembrane (TM) protein Sec61 β-subunit homologue 2 (Sbh2) as a Doa10 substrate. Sbh2 is part of the trimeric Ssh1 complex involved in protein translocation. Unassembled Sbh2 is rapidly degraded in a Doa10-dependent manner. Intriguingly, the degron maps to the Sbh2 TM region. Thus, in contrast to the prevailing view, Doa10 (and presumably its human orthologue) has the capacity for recognizing intramembrane degrons, expanding its spectrum of substrates.
Endoplasmic-reticulum-associated protein degradation
Degron
Ubiquitin-Protein Ligases
Protein Degradation
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Citations (93)