scholarly journals Evolutionarily conserved chaperone-mediated proteasomal degradation of a disease-linked aspartoacylase variant

2020 ◽  
Author(s):  
Sarah K. Gersing ◽  
Yong Wang ◽  
Martin Grønbæk-Thygesen ◽  
Caroline Kampmeyer ◽  
Lene Clausen ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
De Novo ◽  
Neurodegenerative Disorder ◽  
Canavan Disease ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Wild Type ◽  
Spongy Degeneration ◽  
Brain White Matter

AbstractCanavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we examine the stability and degradation pathway of ASPA C152W. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution prevents ASPA from reaching its stable native conformation, presumably by impacting on de novo folding. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. We propose that Hsp110 is a potential therapeutic target for misfolding ASPA variants that trigger Canavan disease due to excessive degradation.

scholarly journals Mapping the degradation pathway of a disease-linked aspartoacylase variant

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009539
Author(s):  
Sarah K. Gersing ◽  
Yong Wang ◽  
Martin Grønbæk-Thygesen ◽  
Caroline Kampmeyer ◽  
Lene Clausen ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Wild Type ◽  
Protein Variant ◽  
Stable Conformation ◽  
Spongy Degeneration ◽  
Brain White Matter

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.

scholarly journals Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation

2015 ◽  
Vol 26 (3) ◽  
pp. 390-405 ◽  
Author(s):  
Abla Tannous ◽  
Nishant Patel ◽  
Taku Tamura ◽  
Daniel N. Hebert
Keyword(s):  
Quality Control ◽  
Secretory Pathway ◽  
Selection Process ◽  
Control Process ◽  
Degradation Pathway ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Mutant Proteins ◽  
Quality Control Process ◽  
Glycoprotein Secretion

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.

scholarly journals Quality Control of a Transcriptional Regulator by SUMO-Targeted Degradation

2009 ◽  
Vol 29 (7) ◽  
pp. 1694-1706 ◽  
Author(s):  
Zheng Wang ◽  
Gregory Prelich
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Physiological Importance ◽  
Sensitive Allele ◽  
Quality Control Mechanism ◽  
Mutant Phenotypes

ABSTRACT Slx5 and Slx8 are heterodimeric RING domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs are proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date is mammalian PML, and the physiological importance of SUMO-targeted ubiquitylation remains largely unknown. We previously identified mutations in SLX5 and SLX8 by selecting for suppressors of a temperature-sensitive allele of MOT1, which encodes a regulator of TATA-binding protein. Here, we demonstrate that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of Saccharomyces cerevisiae in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrate that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.

scholarly journals Mutations Outside the Ure2 Amyloid-Forming Region Disrupt [URE3] Prion Propagation and Alter Interactions with Protein Quality Control Factors

2020 ◽  
Vol 40 (21) ◽  
Author(s):  
Shailesh Kumar ◽  
Elliot A. Dine ◽  
Ethan Paddock ◽  
Danielle N. Steinberg ◽  
Lois E. Greene ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
De Novo ◽  
Protein Quality Control ◽  
Functional Domain ◽  
Amyloid Formation ◽  
Yeast Prions ◽  
Control Factors ◽  
Prion Propagation

ABSTRACT The yeast prion [URE3] propagates as a misfolded amyloid form of the Ure2 protein. Propagation of amyloid-based yeast prions requires protein quality control (PQC) factors, and altering PQC abundance or activity can cure cells of prions. Yeast antiprion systems composed of PQC factors act at normal abundance to restrict establishment of the majority of prion variants that arise de novo. While these systems are well described, how they or other PQC factors interact with prion proteins remains unclear. To gain insight into such interactions, we identified mutations outside the Ure2 prion-determining region that destabilize [URE3]. Despite residing in the functional domain, 16 of 17 mutants retained Ure2 activity. Four characterized mutations caused rapid loss of [URE3] yet allowed [URE3] to propagate under prion-selecting conditions. Two sensitized [URE3] to Btn2, Cur1, and Hsp42, but in different ways. Two others reduced amyloid formation in vitro. Of these, one impaired prion replication and the other apparently impaired transmission. Thus, widely dispersed sites outside a prion’s amyloid-forming region can contribute to prion character, and altering such sites can disrupt prion propagation by altering interactions with PQC factors.

scholarly journals USP5 enhances SGTA mediated protein quality control

2021 ◽  
Author(s):  
Yvonne Nyathi ◽  
Jake Alfie Hill
Keyword(s):  
Quality Control ◽  
Steady State ◽  
Type Ii Diabetes ◽  
Protein Quality ◽  
Proteasomal Degradation ◽  
Aggregate Formation ◽  
Deubiquitinating Enzymes ◽  
Physiological Processes ◽  
Hydrophobic Amino Acids ◽  
Steady State Levels

Mislocalised membrane proteins (MLPs) present a risk to the cell due to exposed hydrophobic amino acids which cause MLPs to aggregate. Previous studies identified SGTA as a key component of the machinery that regulates the quality control of MLPs. Overexpression of SGTA promotes deubiqutination of MLPs resulting in their accumulation in cytosolic inclusions, suggesting SGTA acts in collaboration with deubiquitinating enzymes (DUBs) to exert these effects.  However, the DUBs that play a role in this process have not been identified.  In this study we have identified the ubiquitin specific peptidase 5 (USP5) as a DUB important in regulating the quality control of MLPs. We show that USP5 is in complex with SGTA, and this association is increased in the presence of an MLP. Overexpression of SGTA results in an increase in steady-state levels of MLPs suggesting a delay in proteasomal degradation of substrates. However, our results show that this effect is strongly dependent on the presence of USP5.  We find that in the absence of USP5, the ability of SGTA to increase the steady state levels of MLPs is compromised. Moreover, knockdown of USP5 results in a reduction in the steady state levels of MLPs, while overexpression of USP5 increases the steady state levels. Our findings suggest that the interaction of SGTA with USP5 enables specific MLPs to escape proteasomal degradation allowing selective modulation of MLP quality control. These findings progress our understanding of aggregate formation, a hallmark in a range of neurodegenerative diseases and type II diabetes, as well as physiological processes of aggregate clearance.

scholarly journals Rsp5 Ubiquitin ligase–mediated quality control system clears membrane proteins mistargeted to the vacuole membrane

2018 ◽  
Vol 218 (1) ◽  
pp. 234-250 ◽  
Author(s):  
Richa Sardana ◽  
Lu Zhu ◽  
Scott D. Emr
Keyword(s):  
Quality Control ◽  
Ubiquitin Ligase ◽  
Protein Quality ◽  
De Novo ◽  
Specific Protein ◽  
Cell Integrity ◽  
Vacuole Membrane ◽  
Second Tier ◽  
Safe Mechanism ◽  
Fail Safe

Maintenance of organelle identity is profoundly dependent on the coordination between correct targeting of proteins and removal of mistargeted and damaged proteins. This task is mediated by organelle-specific protein quality control (QC) systems. In yeast, the endocytosis and QC of most plasma membrane (PM) proteins requires the Rsp5 ubiquitin ligase and ART adaptor network. We show that intracellular adaptors of Rsp5, Ear1, and Ssh4 mediate recognition and vacuolar degradation of PM proteins that escape or bypass PM QC systems. This second tier of surveillance helps to maintain cell integrity upon heat stress and protects from proteotoxicity. To understand the mechanism of the recognition of aberrant PM cargos by Ssh4–Rsp5, we mistarget multiple PM proteins de novo to the vacuolar membrane. We found that Ssh4–Rsp5 can target and ubiquitinate multiple lysines within a restricted distance from the membrane, providing a fail-safe mechanism for a diverse cargo repertoire. The mistargeting or misfolding of PM proteins likely exposes these lysines or shifts them into the “ubiquitination zone” accessible to the Ssh4–Rsp5 complex.

Protein folding, misfolding and quality control: the role of molecular chaperones

2014 ◽  
Vol 56 ◽  
pp. 53-68 ◽  
Author(s):  
Katharina Papsdorf ◽  
Klaus Richter
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
De Novo ◽  
Cellular Proteins ◽  
System A ◽  
Protein Quality Control System ◽  
De Novo Folding ◽  
Protective Proteins ◽  
Lateral Sclerosis

Cells have to cope with stressful conditions and adapt to changing environments. Heat stress, heavy metal ions or UV stress induce damage to cellular proteins and disturb the balanced status of the proteome. The adjusted balance between folded and folding proteins, called protein homoeostasis, is required for every aspect of cellular functionality. Protective proteins called chaperones are expressed under extreme conditions in order to prevent aggregation of cellular proteins and safeguard protein quality. These chaperones co-operate during de novo folding, refolding and disaggregation of damaged proteins and in many cases refold them to their functional state. Even under physiological conditions these machines support protein homoeostasis and maintain the balance between de novo folding and degradation. Mutations generating unstable proteins, which are observed in numerous human diseases such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and cystic fibrosis, also challenge the protein quality control system. A better knowledge of how the protein homoeostasis system is regulated will lead to an improved understanding of these diseases and provide potential targets for therapy.

Mitochondrial quality control by the ubiquitin–proteasome system

2011 ◽  
Vol 39 (5) ◽  
pp. 1509-1513 ◽  
Author(s):  
Eric B. Taylor ◽  
Jared Rutter
Keyword(s):  
Quality Control ◽  
Ubiquitin Ligase ◽  
Protein Quality ◽  
Mitochondrial Protein ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Quality Control ◽  
Ubiquitinated Proteins ◽  
Ubiquitin Proteasome

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin–proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.

scholarly journals AAA ATPase p97/Valosin-containing Protein Interacts with gp78, a Ubiquitin Ligase for Endoplasmic Reticulum-associated Degradation

2004 ◽  
Vol 279 (44) ◽  
pp. 45676-45684 ◽  
Author(s):  
Xiaoyan Zhong ◽  
Yuxian Shen ◽  
Petek Ballar ◽  
Andria Apostolou ◽  
Reuven Agami ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Protein Quality ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Aaa Atpase ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Ubiquitin Ligase E3 ◽  
Quality Control Mechanism ◽  
Interacting Domain

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosin-containing protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3δ, gp78 consistently enhances p97/VCP-CD3δ binding and facilitates CD3δ degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3δ, and abrogates VCP-CD3δ interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3δ degradation and leads to accumulation of polyubiquitinated CD3δ, suggesting a failure in delivering ubiquitinated CD3δ for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.

scholarly journals Proteotoxic stress promotes entrapment of ribosomes and misfolded proteins in a shared cytosolic compartment

2020 ◽  
Vol 48 (7) ◽  
pp. 3888-3905
Author(s):  
Arnab Ghosh ◽  
Loren Dean Williams ◽  
Dimitri G Pestov ◽  
Natalia Shcherbik
Keyword(s):  
Quality Control ◽  
Protein Synthesis ◽  
Cell Viability ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Active Duty ◽  
Misfolded Proteins ◽  
Wild Type ◽  
Peak Efficiency ◽  
Proteotoxic Stress

Abstract Cells continuously monitor protein synthesis to prevent accumulation of aberrant polypeptides. Insufficient capacity of cellular degradative systems, chaperone shortage or high levels of mistranslation by ribosomes can result in proteotoxic stress and endanger proteostasis. One of the least explored reasons for mistranslation is the incorrect functioning of the ribosome itself. To understand how cells deal with ribosome malfunction, we introduced mutations in the Expansion Segment 7 (ES7L) of 25S rRNA that allowed the formation of mature, translationally active ribosomes but induced proteotoxic stress and compromised cell viability. The ES7L-mutated ribosomes escaped nonfunctional rRNA Decay (NRD) and remained stable. Remarkably, ES7L-mutated ribosomes showed increased segregation into cytoplasmic foci containing soluble misfolded proteins. This ribosome entrapment pathway, termed TRAP (Translational Relocalization with Aberrant Polypeptides), was generalizable beyond the ES7L mutation, as wild-type ribosomes also showed increased relocalization into the same compartments in cells exposed to proteotoxic stressors. We propose that during TRAP, assembled ribosomes associated with misfolded nascent chains move into cytoplasmic compartments enriched in factors that facilitate protein quality control. In addition, TRAP may help to keep translation at its peak efficiency by preventing malfunctioning ribosomes from active duty in translation.

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