Genetic analysis reveals functions of atypical polyubiquitin chains

Gutiérrez, Fernando; Şimşek, Deniz; Mizrak, Arda; Deutschbauer, Adam M.; Braberg, Hannes; Johnson, Jeffrey R.; Xu, Jiewei; Shales, Michael; Nguyen, Michelle; Tamse-Kuehn, Raquel; Palm, Curt; Steinmetz, Lars M.; Krogan, Nevan J.; Toczyski, David P.

doi:10.7554/elife.42955

Cited by 12 publications

(13 citation statements)

References 108 publications

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“…This may explain why SHANK, MOV10 and IkB were not identified as significant targets of K48 in either sex during fear memory formation in our dataset, despite previous evidence demonstrating proteasome-mediated degradation of these proteins following learning (Lopez-Salon et al, 2001 ; Yeh et al, 2002 ; Lee et al, 2008 ; Jarome et al, 2011 ). Furthermore, this could have occurred due to these putative degradation targets being marked by a polyubiquitin chain carrying another linkage site, such as K11 (Xu et al, 2009 ; Meyer and Rape, 2014 ; Meza Gutierrez et al, 2018 ; Musaus et al, 2020 ). Unfortunately, until more precise technology is developed, the specific role of K48 polyubiquitination in fear memory formation in any brain region will remain equivocal.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory Formation

Farrell

Musaus

Navabpour

et al. 2021

Front. Mol. Neurosci.

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Ubiquitin-proteasome mediated protein degradation has been widely implicated in fear memory formation in the amygdala. However, to date, the protein targets of the proteasome remain largely unknown, limiting our understanding of the functional significance for protein degradation in fear memory formation. Additionally, whether similar proteins are targeted by the proteasome between sexes has yet to be explored. Here, we combined a degradation-specific K48 Tandem Ubiquitin Binding Entity (TUBE) with liquid chromatography mass spectrometry (LC/MS) to identify the target substrates of the protein degradation process in the amygdala of male and female rats following contextual fear conditioning. We found that males (43) and females (77) differed in the total number of proteins that had significant changes in K48 polyubiquitin targeting in the amygdala following fear conditioning. Many of the identified proteins (106) had significantly reduced levels in the K48-purified samples 1 h after fear conditioning, suggesting active degradation of the substrate due to learning. Interestingly, only 3 proteins overlapped between sexes, suggesting that targets of the protein degradation process may be sex-specific. In females, many proteins with altered abundance in the K48-purified samples were involved in vesicle transport or are associated with microtubules. Conversely, in males, proteins involved in the cytoskeleton, ATP synthesis and cell signaling were found to have significantly altered abundance. Only 1 protein had an opposite directional change in abundance between sexes, LENG1, which was significantly enhanced in males while lower in females. This suggests a more rapid degradation of this protein in females during fear memory formation. Interestingly, GFAP, a critical component of astrocyte structure, was a target of K48 polyubiquitination in both males and females, indicating that protein degradation is likely occurring in astrocytes following fear conditioning. Western blot assays revealed reduced levels of these target substrates following fear conditioning in both sexes, confirming that the K48 polyubiquitin was targeting these proteins for degradation. Collectively, this study provides strong evidence that sex differences exist in the protein targets of the degradation process in the amygdala following fear conditioning and critical information regarding how ubiquitin-proteasome mediated protein degradation may contribute to fear memory formation in the brain.

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Section: Discussionmentioning

confidence: 99%

Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory Formation

Farrell

Musaus

Navabpour

et al. 2021

Front. Mol. Neurosci.

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“…Lastly, we affinity purified 26S particles (with or without MG132 treatment) from yeast strains designed to individually replace K11, K48 and K63 with arginine, thus preventing formation of Ub-Ub linkages through those sites (Meza-Gutierrez et al, 2018). Whereas the K11R and K63R strains replaced all Ub coding regions with K-to-R variants, the K48R strain replaced only ∼80% of wild-type Ub, due to the essential nature of K48-linked chains (Meza-Gutierrez et al, 2018). When inhibited proteasomes were affinity purified from these strains using the Pre1-TEV-ProA tag, only the K11R strain retained robust proteasome ubiquitylation, while those for the K48R and K63R strains were substantially suppressed (Figure 7C).…”

Section: Resultsmentioning

confidence: 99%

“…Given that assembly of K48- and K63-linked Ub chains might provide a critical signal for the proteaphagy of dysfunctional particles, we tested whether yeast unable to make such polymers were also compromised in proteasome clearance. Here, we introduced the Pre10-GFP and Rpn5-GFP reporters into the complete library of K-to-R Ub substitutions (Meza-Gutierrez et al, 2018), and measured proteaphagy by the free GFP release assay and/or microscopy. When all seven K-to-R variants were assayed for free GFP after pre-treating the cells with MG132, only the K48R and K63R variants markedly slowed proteaphagy (Figure 7D).…”

Section: Resultsmentioning

confidence: 99%

A Trio of Ubiquitin Ligases Sequentially Drive Ubiquitylation and Autophagic Degradation of Dysfunctional Yeast Proteasomes

2021

Preprint

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As central effectors of ubiquitin (Ub)-mediated proteolysis, proteasomes are regulated at multiple levels, including degradation of unwanted or dysfunctional particles via autophagy (termed proteaphagy). In yeast, inactive proteasomes are exported from the nucleus, sequestered into cytoplasmic aggresomes via the Hsp42 chaperone, extensively ubiquitylated, and then tethered to the expanding phagophore by the autophagy receptor Cue5. Here, we demonstrate the need for ubiquitylation driven by the trio of Ub ligases (E3s) San1, Rsp5 and Hul5, which, together with their corresponding E2s, work sequentially to promote nuclear export and Cue5 recognition. Whereas San1 functions prior to nuclear export, Rsp5 and Hul5 likely decorate aggresome-localized proteasomes in concert. Ultimately, topologically complex Ub chain(s) containing both K48 and K63 Ub-Ub linkages are assembled, mainly on the regulatory particle, to generate autophagy-competent substrates. As San1, Rsp5, Hul5, Hsp42, and Cue5 also participate in general proteostasis, proteaphagy likely engages an essential mechanism for eliminating inactive/misfolded proteins.

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“…Ubiquitin has seven lysine residues (K6, K11, K27, K29, K33, K48 and K63), so eight structurally distinct types of poly-ubiquitin chain linkage can be formed, together with the N-terminal methionine (M1; a head-to-tail linear linkage). The anaphase-promoting complex (APC/C) can assemble K11-linked and K48-linked ubiquitin chains on substrates 38– 45 . In order to build K11-linked ubiquitin chains, the metazoan APC/C uses two families of E2 enzymes: a “chain-initiating” E2 such as Ube2C/UbcH10 and Ube2D/UbcH5 and an “elongating” E2 such as Ube2S 38– 40, 46, 47 .…”

Section: The Apc/c Employs Two E2s and Assembles Poly-ubiquitin Chainsmentioning

confidence: 99%

APC/C: current understanding and future perspectives

Yamano

2019

F1000Res

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The separation of sister chromatids at anaphase, which is regulated by an E3 ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C), is arguably the most important irrevocable event during the cell cycle. The APC/C and cyclin-dependent kinase 1 (Cdk1) are just two of the many significant cell cycle regulators and exert control through ubiquitylation and phosphorylation, respectively. The temporal and spatial regulation of the APC/C is achieved by multiple mechanisms, including phosphorylation, interaction with the structurally related co-activators Cdc20 and Cdh1, loading of distinct E2 ubiquitin-conjugating enzymes, binding with inhibitors and differential affinities for various substrates. Since the discovery of APC/C 25 years ago, intensive studies have uncovered many aspects of APC/C regulation, but we are still far from a full understanding of this important cellular machinery. Recent high-resolution cryogenic electron microscopy analysis and reconstitution of the APC/C have greatly advanced our understanding of molecular mechanisms underpinning the enzymatic properties of APC/C. In this review, we will examine the historical background and current understanding of APC/C regulation.

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Genetic analysis reveals functions of atypical polyubiquitin chains

Cited by 12 publications

References 108 publications

Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory Formation

Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory Formation

A Trio of Ubiquitin Ligases Sequentially Drive Ubiquitylation and Autophagic Degradation of Dysfunctional Yeast Proteasomes

APC/C: current understanding and future perspectives

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