scholarly journals Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mary D. Cundiff ◽  
Christina M. Hurley ◽  
Jeremy D. Wong ◽  
Joseph A. Boscia ◽  
Aarti Bashyal ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Motor Proteins ◽  
Ubiquitin Proteasome System ◽  
Physical Models ◽  
Protein Domain ◽  
Eukaryotic Cells ◽  
Multiple Receptors ◽  
Ubiquitin Proteasome ◽  
Ubiquitin Receptors ◽  
Partial Redundancy

Abstract The ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasome’s unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

scholarly journals Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

2019 ◽  
Author(s):  
Mary D. Cundiff ◽  
Christina M. Hurley ◽  
Jeremy D. Wong ◽  
Aarti Bashyal ◽  
Jake Rosenberg ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Motor Proteins ◽  
Ubiquitin Proteasome System ◽  
Physical Models ◽  
Protein Domain ◽  
Eukaryotic Cells ◽  
Multiple Receptors ◽  
Ubiquitin Proteasome ◽  
Ubiquitin Receptors ◽  
Partial Redundancy

ABSTRACTThe ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasome’s unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

scholarly journals Mode of Targeting to the Proteasome Determines GFP Fate

2020 ◽  
Author(s):  
Christopher E. Bragança ◽  
Daniel A. Kraut
Keyword(s):  
Fluorescent Protein ◽  
Ubiquitin Proteasome System ◽  
Eukaryotic Cells ◽  
Polyubiquitin Chains ◽  
Ubiquitin Proteasome ◽  
Targeting Signal ◽  
Green Fluorescent ◽  
Ubiquitin Receptors ◽  
Multiple Variants ◽  
Domain Stability

ABSTRACTThe Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different stabilities. We previously found that the proteasome’s ability to unfold and degrade substrates is enhanced by polyubiquitin chains on the substrate, and that proteasomal ubiquitin receptors mediate this activation. Herein we investigate how the fate of GFP variants of differing stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates.

scholarly journals Mode of targeting to the proteasome determines GFP fate

2020 ◽  
Vol 295 (47) ◽  
pp. 15892-15901
Author(s):  
Christopher Eric Bragança ◽  
Daniel Adam Kraut
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Canonical Pathway ◽  
Eukaryotic Cells ◽  
Reporter Protein ◽  
Ubiquitin Proteasome ◽  
Targeting Signal ◽  
Multiple Variants ◽  
Domain Stability

The ubiquitin–proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, whereas the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.

scholarly journals Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

2020 ◽  
Author(s):  
Ganapathi Kandasamy ◽  
Ashis Kumar Pradhan ◽  
R Palanimurugan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Rna Degradation ◽  
26S Proteasome ◽  
Cellular Proteins ◽  
Cellular Homeostasis ◽  
Substrate Degradation ◽  
Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

scholarly journals E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

Oncogene ◽  
2006 ◽  
Vol 26 (3) ◽  
pp. 441-448 ◽  
Author(s):  
L Sun ◽  
J S Trausch-Azar ◽  
A Ciechanover ◽  
A L Schwartz
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Muscle Differentiation ◽  
Ubiquitin Proteasome

scholarly journals Involvement of the Ubiquitin-Proteasome System in the Formation of Experimental Postsurgical Peritoneal Adhesions

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Clara Di Filippo ◽  
Pasquale Petronella ◽  
Fulvio Freda ◽  
Marco Scorzelli ◽  
Marco Ferretti ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
20S Proteasome ◽  
Intracellular Protein ◽  
Peritoneal Adhesions ◽  
Proteasome Subunit ◽  
Peritoneal Tissue ◽  
Intracellular Protein Degradation ◽  
Ubiquitin Proteasome

We investigated the Ubiquitin-Proteasome System (UPS), major nonlysosomal intracellular protein degradation system, in the genesis of experimental postsurgical peritoneal adhesions. We assayed the levels of UPS within the adhered tissue along with the development of peritoneal adhesions and used the specific UPS inhibitor bortezomib in order to assess the effect of the UPS blockade on the peritoneal adhesions. We found a number of severe postsurgical peritoneal adhesions at day 5 after surgery increasing until day 10. In the adhered tissue an increased values of ubiquitin and the 20S proteasome subunit, NFkB, IL-6, TNF-αand decreased values of IkB-beta were found. In contrast, bortezomib-treated rats showed a decreased number of peritoneal adhesions, decreased values of ubiquitin and the 20S proteasome, NFkB, IL-6, TNF-α, and increased levels of IkB-beta in the adhered peritoneal tissue. The UPS system, therefore, is primarily involved in the formation of post-surgical peritoneal adhesions in rats.

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