Sequestosome 1/p62 Is a Polyubiquitin Chain Binding Protein Involved in Ubiquitin Proteasome Degradation

ABSTRACT Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.

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Structural insights into ubiquitin recognition and Ufd1 interaction of Npl4

Nature Communications ◽

10.1038/s41467-019-13697-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Yusuke Sato ◽

Hikaru Tsuchiya ◽

Atsushi Yamagata ◽

Kei Okatsu ◽

Keiji Tanaka ◽

...

Keyword(s):

Crystal Structures ◽

Mutational Analysis ◽

Proteasomal Degradation ◽

Degradation Pathway ◽

Binding Motif ◽

Polyubiquitin Chain ◽

Hydrophobic Groove ◽

Terminal Loop ◽

Initial Binding ◽

Structural Insights

AbstractNpl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48–UN complex and its assembly.

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Human Fas-Associated Factor 1, Interacting with Ubiquitinated Proteins and Valosin-Containing Protein, Is Involved in the Ubiquitin-Proteasome Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.25.6.2511-2524.2005 ◽

2005 ◽

Vol 25 (6) ◽

pp. 2511-2524 ◽

Cited By ~ 73

Author(s):

Eun Joo Song ◽

Seung-Hee Yim ◽

Eunhee Kim ◽

Nam-Soon Kim ◽

Kong-Joo Lee

Keyword(s):

Scaffolding Protein ◽

Cellular Functions ◽

Associated Factor ◽

Binding Partner ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitinated Proteins ◽

Uba Domain ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Novel Protein

ABSTRACT Human Fas-associated factor 1 (hFAF1) is a novel protein having multiubiquitin-related domains. We investigated the cellular functions of hFAF1 and found that valosin-containing protein (VCP), the multiubiquitin chain-targeting factor in the degradation of the ubiquitin-proteasome pathway, is a binding partner of hFAF1. hFAF1 is associated with the ubiquitinated proteins via the newly identified N-terminal UBA domain and with VCP via the C-terminal UBX domain. The overexpression of hFAF1 and a truncated UBA domain inhibited the degradation of ubiquitinated proteins and increased cell death. These results suggest that hFAF1 binding to ubiquitinated protein and VCP is involved in the ubiquitin-proteasome pathway. We hypothesize that hFAF1 may serve as a scaffolding protein that regulates protein degradation in the ubiquitin-proteasome pathway.

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Negative Regulator of Ubiquitin-Like Protein 1 modulates the autophagy–lysosomal pathway via p62 to facilitate the extracellular release of tau following proteasome impairment

Human Molecular Genetics ◽

10.1093/hmg/ddz255 ◽

2019 ◽

Vol 29 (1) ◽

pp. 80-96 ◽

Cited By ~ 3

Author(s):

Rosellina Guarascio ◽

Dervis Salih ◽

Marina Yasvoina ◽

Frances A Edwards ◽

Michael E Cheetham ◽

...

Keyword(s):

Hippocampal Neurons ◽

Proteasome Inhibition ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Tau Phosphorylation ◽

Negative Regulator ◽

Autophagy Flux ◽

Extracellular Release ◽

Uba Domain ◽

Ubiquitin Proteasome

Abstract Negative regulator of ubiquitin-like protein 1 (NUB1) and its longer isoform NUB1L are ubiquitin-like (UBL)/ubiquitin-associated (UBA) proteins that facilitate the targeting of proteasomal substrates, including tau, synphilin-1 and huntingtin. Previous data revealed that NUB1 also mediated a reduction in tau phosphorylation and aggregation following proteasome inhibition, suggesting a switch in NUB1 function from targeted proteasomal degradation to a role in autophagy. Here, we delineate the mechanisms of this switch and show that NUB1 interacted specifically with p62 and induced an increase in p62 levels in a manner facilitated by inhibition of the proteasome. NUB1 moreover increased autophagosomes and the recruitment of lysosomes to aggresomes following proteasome inhibition. Autophagy flux assays revealed that NUB1 affected the autophagy–lysosomal pathway primarily via the UBA domain. NUB1 localized to cytosolic inclusions with pathological forms of tau, as well as LAMP1 and p62 in the hippocampal neurons of tauopathy mice. Finally, NUB1 facilitated the extracellular release of tau following proteasome inhibition. This study thus shows that NUB1 plays a role in regulating the autophagy–lysosomal pathway when the ubiquitin proteasome system is compromised, thus contributing to the mechanisms targeting the removal of aggregation-prone proteins upon proteasomal impairment.

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Disruption of ubiquitin-mediated processes in diseases of the brain and bone

Biochemical Society Transactions ◽

10.1042/bst0360469 ◽

2008 ◽

Vol 36 (3) ◽

pp. 469-471 ◽

Cited By ~ 8

Author(s):

Robert Layfield ◽

Mark S. Searle

Keyword(s):

Disease Process ◽

Protein Aggregates ◽

Ubiquitin Proteasome System ◽

Nuclear Factor Κb ◽

Negative Effects ◽

Multifunctional Protein ◽

Sequestosome 1 ◽

Ubiquitin Binding ◽

Uba Domain ◽

Ubiquitin Proteasome

A role for ubiquitin in the pathogenesis of human diseases was first suggested some two decades ago, from studies that localized the protein to intracellular protein aggregates, which are a feature of the major human neurodegenerative disorders. Although several different mechanisms have been proposed to connect impairment of the UPS (ubiquitin–proteasome system) to the presence of these ‘ubiquitin inclusions’ within diseased neurones, their significance in the disease process remains to be fully clarified. Ubiquitin inclusions also contain ubiquitin-binding proteins, such as the p62 protein [also known as SQSTM1 (sequestosome 1)], which non-covalently interacts with the ubiquitinated protein aggregates and may serve to mediate their autophagic clearance. p62 is a multifunctional protein and, in the context of bone-resorbing osteoclasts, is an important scaffold in the RANK [receptor activator of NF-κB (nuclear factor κB)]–NF-κB signalling pathway. Further, mutations affecting the UBA domain (ubiquitin-associated domain) of p62 are commonly found in patients with the skeletal disorder PDB (Paget's disease of bone). These mutations impair the ability of p62 to bind to ubiquitin and result in disordered osteoclast NF-κB signalling that may underlie the disease aetiology. Recent structural insights into the unusual mechanism of ubiquitin recognition by the p62 UBA domain have helped rationalize the mechanisms by which different PDB mutations exert their negative effects on ubiquitin binding by p62, as well as providing an indication of the ubiquitin-binding selectivity of p62 and, by extension, its normal biological functions.

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146: Clusterin is a Ubiquitin Binding Protein Involved in Murr1 and 1-KB Proteasomal Degradation to Confer Cell Survival in Prostate Cancer

The Journal of Urology ◽

10.1016/s0022-5347(18)30411-7 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 50-50

Author(s):

Amina Zoubeidi ◽

Eliana Beraldi ◽

Anousheh Zardan ◽

Martin E. Gleave

Keyword(s):

Prostate Cancer ◽

Cell Survival ◽

Binding Protein ◽

Proteasomal Degradation ◽

Ubiquitin Binding

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Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e06-02-0106 ◽

2006 ◽

Vol 17 (7) ◽

pp. 3281-3290 ◽

Cited By ~ 23

Author(s):

Jing Xiao ◽

Leslie S. Kim ◽

Todd R. Graham

Keyword(s):

Atpase Activity ◽

Point Mutation ◽

Mutational Analysis ◽

Weak Interactions ◽

Tetratricopeptide Repeat ◽

Uba Domain ◽

J Domain ◽

Tpr Domain

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

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Role of Ubiquitin Ligases and the Proteasome in Oncogenesis: Novel Targets for Anticancer Therapies

Journal of Clinical Oncology ◽

10.1200/jco.2012.44.0958 ◽

2013 ◽

Vol 31 (9) ◽

pp. 1231-1238 ◽

Cited By ~ 100

Author(s):

Lindsey N. Micel ◽

John J. Tentler ◽

Peter G. Smith ◽

Gail S. Eckhardt

Keyword(s):

Cell Cycle ◽

Anticancer Agents ◽

Cell Cycle Control ◽

Ubiquitin Proteasome System ◽

Cellular Regulation ◽

Ubiquitin Chain ◽

Key Enzyme ◽

Ubiquitin Proteasome ◽

Enzyme Families ◽

And Function

The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to cellular regulation and function. The UPS comprises a sequential series of enzymatic processes using four key enzyme families: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-carrier proteins), E3 (ubiquitin-protein ligases), and E4 (ubiquitin chain assembly factors). Because the UPS is a crucial regulator of the cell cycle, and abnormal cell-cycle control can lead to oncogenesis, aberrancies within the UPS pathway can result in a malignant cellular phenotype and thus has become an attractive target for novel anticancer agents. This article will provide an overall review of the mechanics of the UPS, describe aberrancies leading to cancer, and give an overview of current drug therapies selectively targeting the UPS.

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

10.1101/2020.03.30.015370 ◽

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.

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NBR1 directly promotes the formation of p62 – ubiquitin condensates via its PB1 and UBA domains

10.1101/2020.09.18.303552 ◽

2020 ◽

Author(s):

Adriana Savova ◽

Julia Romanov ◽

Sascha Martens

Keyword(s):

Phase Separation ◽

Wild Type ◽

Selective Autophagy ◽

Ubiquitinated Proteins ◽

Cargo Receptor ◽

Condensate Formation ◽

Uba Domain ◽

Reconstituted System ◽

Pb1 Domain

SummarySelective autophagy removes harmful intracellular structures such as ubiquitinated, aggregated proteins ensuring cellular homeostasis. This is achieved by the encapsulation of this cargo material within autophagosomes. The cargo receptor p62/SQSTM1 mediates the phase separation of ubiquitinated proteins into condensates, which subsequently become targets for the autophagy machinery. NBR1, another cargo receptor, is a crucial regulator of condensate formation. The mechanisms of the interplay between p62 and NBR1 are not well understood. Employing a fully reconstituted system we show that two domains of NBR1, the PB1 domain which binds to p62 and the UBA domain which binds to ubiquitin, are required to promote p62-ubiquitin condensate formation. In cells, acute depletion of endogenous NBR1 reduces formation of p62 condensates, a phenotype that can be rescued by re-expression of wild-type NBR1, but not PB1 or UBA domain mutants. Our results provide mechanistic insights into the role of NBR1 in selective autophagy.

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