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 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 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.

scholarly journals Recognition of enzymes lacking bound cofactor by protein quality control

2016 ◽  
Vol 113 (43) ◽  
pp. 12156-12161 ◽  
Author(s):  
Adrián Martínez-Limón ◽  
Marion Alriquet ◽  
Wei-Han Lang ◽  
Giulia Calloni ◽  
Ilka Wittig ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Wild Type ◽  
Interacting Protein ◽  
Cofactor Binding ◽  
Enzymatic Function ◽  
Protein Biogenesis ◽  
Related Enzyme

Protein biogenesis is tightly linked to protein quality control (PQC). The role of PQC machinery in recognizing faulty polypeptides is becoming increasingly understood. Molecular chaperones and cytosolic and vacuolar degradation systems collaborate to detect, repair, or hydrolyze mutant, damaged, and mislocalized proteins. On the other hand, the contribution of PQC to cofactor binding-related enzyme maturation remains largely unexplored, although the loading of a cofactor represents an all-or-nothing transition in regard to the enzymatic function and thus must be surveyed carefully. Combining proteomics and biochemical analysis, we demonstrate here that cells are able to detect functionally immature wild-type enzymes. We show that PQC-dedicated ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP) recognizes and marks for degradation not only a mutant protein but also its wild-type variant as long as the latter remains cofactor free. A distinct structural feature, the protruding C-terminal tail, which appears in both the mutant and wild-type polypeptides, contributes to recognition by CHIP. Our data suggest that relative insufficiency of apoprotein degradation caused by cofactor shortage can increase amyloidogenesis and aggravate protein aggregation disorders.

scholarly journals The extent of Ssa1/Ssa2 Hsp70 chaperone involvement in nuclear protein quality control degradation varies with the substrate

2020 ◽  
Vol 31 (3) ◽  
pp. 221-233 ◽  
Author(s):  
Ramon D. Jones ◽  
Charisma Enam ◽  
Rebeca Ibarra ◽  
Heather R. Borror ◽  
Kaitlyn E. Mostoller ◽  
...  
Keyword(s):  
Quality Control ◽  
Nuclear Protein ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Proteasomal Degradation ◽  
Misfolded Proteins ◽  
Hsp70 Chaperone

The ubiquitin-mediated proteasomal degradation of misfolded proteins is generally thought to require Hsp70 chaperones, particularly Ssa1 and Ssa2 in yeast. This study reveals that Ssa1/Ssa2 are involved in the degradation of misfolded proteins in the yeast nucleus, but their degree of involvement varies depending on the misfolded nuclear protein.

scholarly journals Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Jacob A. Culver ◽  
Malaiyalam Mariappan
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Proteasomal Degradation ◽  
Transmembrane Domains ◽  
Misfolded Proteins ◽  
Hydrophobic Membrane ◽  
Cytosolic Protein ◽  
Ta Proteins

Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.

scholarly journals Stat2 stability regulation: an intersection between immunity and carcinogenesis

2020 ◽  
Vol 52 (9) ◽  
pp. 1526-1536 ◽  
Author(s):  
Cheol-Jung Lee ◽  
Hyun-Jung An ◽  
Eun Suh Cho ◽  
Han Chang Kang ◽  
Joo Young Lee ◽  
...  
Keyword(s):  
Quality Control ◽  
Immune Responses ◽  
Molecular Mechanisms ◽  
Protein Quality ◽  
Viral Infections ◽  
Human Cancer ◽  
Protein Quality Control ◽  
Degradation Pathway ◽  
Protein Activity ◽  
Inflammatory Reactions

Abstract Signal transducer and activator of transcription (STAT2) is a member of the STAT family that plays an essential role in immune responses to extracellular and intracellular stimuli, including inflammatory reactions, invasion of foreign materials, and cancer initiation. Although the majority of STAT2 studies in the last few decades have focused on interferon (IFN)-α/β (IFNα/β) signaling pathway-mediated host defense against viral infections, recent studies have revealed that STAT2 also plays an important role in human cancer development. Notably, strategic research on STAT2 function has provided evidence that transient regulatory activity by homo- or heterodimerization induces its nuclear localization where it to forms a ternary IFN-stimulated gene factor 3 (ISGF3) complex, which is composed of STAT1 and/or STAT2 and IFN regulatory factor 9 (IEF9). The molecular mechanisms of ISGF3-mediated ISG gene expression provide the basic foundation for the regulation of STAT2 protein activity but not protein quality control. Recently, previously unknown molecular mechanisms of STAT2-mediated cell proliferation via STAT2 protein quality control were elucidated. In this review, we briefly summarize the role of STAT2 in immune responses and carcinogenesis with respect to the molecular mechanisms of STAT2 stability regulation via the proteasomal degradation pathway.

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