A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.

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Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽

10.3390/biom11010110 ◽

2021 ◽

Vol 11 (1) ◽

pp. 110

Author(s):

Dina Aweida ◽

Shenhav Cohen

Keyword(s):

Protein Quality ◽

Protein Structures ◽

Ubiquitin Proteasome System ◽

Surface Proteins ◽

Catalytic Core ◽

Complex Protein ◽

Ubiquitin Proteasome ◽

Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

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HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.23.16.5790-5802.2003 ◽

2003 ◽

Vol 23 (16) ◽

pp. 5790-5802 ◽

Cited By ~ 225

Author(s):

Arnaud Parcellier ◽

Elise Schmitt ◽

Sandeep Gurbuxani ◽

Daphné Seigneurin-Berny ◽

Alena Pance ◽

...

Keyword(s):

Direct Interaction ◽

Cell Types ◽

26S Proteasome ◽

Necrosis Factor Alpha ◽

Polyubiquitin Chains ◽

Ubiquitinated Proteins ◽

Activation Of Transcription ◽

Ubiquitin Proteasome

ABSTRACT HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.

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Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

Biochemical Journal ◽

10.1042/bj20112175 ◽

2012 ◽

Vol 443 (3) ◽

pp. 681-689 ◽

Cited By ~ 15

Author(s):

Wan Ning Vanessa Chow ◽

Hon Wing Luk ◽

Ho Yin Edwin Chan ◽

Kwok-Fai Lau

Keyword(s):

Cell Death ◽

Degradation Mechanism ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Adaptor Protein ◽

Ubiquitin Proteasome System ◽

Exon 1 ◽

Ubiquitin Proteasome

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW–polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.

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Quality Control of a Transcriptional Regulator by SUMO-Targeted Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.01470-08 ◽

2009 ◽

Vol 29 (7) ◽

pp. 1694-1706 ◽

Cited By ~ 55

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.

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Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

Molecular Biology of the Cell ◽

10.1091/mbc.e15-02-0102 ◽

2015 ◽

Vol 26 (24) ◽

pp. 4325-4332 ◽

Cited By ~ 33

Author(s):

Mingwei Min ◽

Tycho E. T. Mevissen ◽

Maria De Luca ◽

David Komander ◽

Catherine Lindon

Keyword(s):

Cell Cycle Progression ◽

Degradation Kinetics ◽

Ubiquitin Proteasome System ◽

Mitotic Exit ◽

Anaphase Promoting Complex ◽

Polyubiquitin Chains ◽

Substrate Degradation ◽

Ubiquitin Proteasome ◽

In Cells

The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.

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Functional expression and localisation of HOPS/TMUB1 in mouse lens

Bioscience Reports ◽

10.1042/bsr20203998 ◽

2021 ◽

Vol 41 (2) ◽

Author(s):

Daniela Bartoli ◽

Danilo Piobbico ◽

Marilena Castelli ◽

Stefania Pieroni ◽

Damiano Scopetti ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Functional Expression ◽

Transitional Zone ◽

Structural Function ◽

Ubiquitin Proteasome ◽

Lens Cell ◽

Mouse Lens ◽

Functional Phenotype

Abstract Transparency represents the functional phenotype of eye lens. A number of defined steps including quiescence, proliferation, migration and cell differentiation culminates in cell elongation and organelle degradation, allowing the light to reach the retina. HOPS (Hepatocyte Odd Protein Shuttling)/TMUB1 (Trans Membrane Ubiquitin-like containing protein 1) is a nucleo-cytoplasmic shuttling protein, highly expressed both in vivo and in vitro proliferating systems, bearing a ubiquitin-like domain. The present study shows HOPS expression during the phases of lens cell proliferation and fiber differentiation, and its localisation in lens compartments. In lens, HOPS localises mainly in the nucleus of central epithelial cells. During mitosis, HOPS/TMUB1 shuttles to the cytoplasm and returns to the nucleus at the end of mitosis. The differentiating cells share distinct HOPS/TMUB1 localisation in transitional zone depending on the differentiation phases. HOPS/TMUB1 is observed in lens cortex and nucleus. Here, it is attached to fibers, having a structural function with crystallin proteins, probably acting in the ubiquitin–proteasome system.

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Bifunctional Small Molecules That Mediate the Degradation of Extracellular Proteins

10.26434/chemrxiv.12732689.v1 ◽

2020 ◽

Author(s):

David Caianiello ◽

Mengwen Zhang ◽

Jason Ray ◽

Jake Swartzel ◽

Emily Branham ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Extracellular Proteins ◽

Target Protein ◽

Disease Treatment ◽

Bifunctional Molecules ◽

Synthetic Molecules ◽

Ubiquitin Proteasome ◽

Mode A

Targeted protein degradation (TPD) has emerged as a promising and exciting therapeutic strategy. The majority of existing TPD technologies rely on the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modularly designed, bifunctional synthetic molecules called MoDE-As (Molecular Degraders of Extracellular proteins through the Asialoglycoprotein receptor (ASGPR)), which are capable of mediating the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and the ASGPR, which is expressed primarily on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing bifunctional molecules that induce the degradation of both antibody and pro-inflammatory cytokine proteins. To our knowledge, these data represent the first experimental evidence that non-proteinogenic, synthetic molecules can be employed for the TPD of extracellular proteins both in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

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β-TrCP-mediated ubiquitination and degradation of Dlg5 regulates hepatocellular carcinoma cell proliferation

Cancer Cell International ◽

10.1186/s12935-019-1029-1 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Dongping Wang ◽

Qi Zhang ◽

Fenfen Li ◽

Chan Wang ◽

Changming Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Xenograft Model ◽

Ubiquitin Proteasome System ◽

Tumor Xenograft ◽

Dependent Manner ◽

Ubiquitin Proteasome ◽

Novel Strategy ◽

Hcc Cells

Abstract Background Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase (MAGUK) adaptor family of proteins and its deregulation has been implicated in the malignancy of several cancer types. Dlg5 was down-regulated in hepatocellular carcinoma (HCC) and lower Dlg5 expression was associated with poor survival of HCC patients. However, how to regulate Dlg5 remains largely unknown. Methods The co-immunoprecipitation assay was used to determine the interaction between Dlg5 and β-TrCP. The in vivo ubiquitination assay was performed to determine the regulation of Dlg5 by β-TrCP. CCK-8 and colony formation assay were implemented to detect the biological effect of Dlg5 on the growth of HCC cells in vitro. The effect of Dlg5 on HCC tumor growth in vivo was studied in a tumor xenograft model in mice. Results Here we report that Dlg5 is regulated by the ubiquitin proteasome system and depletion of either Cullin 1 or β-TrCP led to increased levels of Dlg5. β-TrCP regulated Dlg5 protein stability by targeting it for ubiquitination and subsequent destruction in a phosphorylation-dependent manner. We further demonstrated a crucial role of Ser730 in the non-canonical phosphodegron of Dlg5 in governing β-TrCP-mediated Dlg5 degradation. Importantly, failure to degrade Dlg5 significantly inhibited HCC cells proliferation both in vitro and in vivo. Conclusion Collectively, our finding provides a novel molecular mechanism for the negative regulation of Dlg5 by β-TRCP in HCC cells. It further suggests that preventing Dlg5 degradation could be a possible novel strategy for clinical treatment of HCC.

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Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation

Biochemical Journal ◽

10.1042/bj20120542 ◽

2012 ◽

Vol 448 (1) ◽

pp. 55-65 ◽

Cited By ~ 19

Author(s):

Jonas Boehringer ◽

Christiane Riedinger ◽

Konstantinos Paraskevopoulos ◽

Eachan O. D. Johnson ◽

Edward D. Lowe ◽

...

Keyword(s):

Protein Interactions ◽

Functional Characterization ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Structural Motif ◽

Protein Protein Interactions ◽

Ubiquitin Proteasome

The ubiquitin–proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein–protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

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Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration

Journal of Biological Chemistry ◽

10.1074/jbc.m116.733626 ◽

2016 ◽

Vol 291 (33) ◽

pp. 17209-17227 ◽

Cited By ~ 32

Author(s):

Dolores Del Prete ◽

Richard C. Rice ◽

Anjali M. Rajadhyaksha ◽

Luciano D'Adamio

Keyword(s):

Amyloid Precursor Protein ◽

Transmitter Release ◽

Ubiquitin Proteasome System ◽

Precursor Protein ◽

E3 Ligases ◽

Ubiquitin Proteasome ◽

Functional Pathway ◽

Related Proteins

The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4CRBN, which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4CRBN, pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys649/650/651/676/688. Deletion of Crbn reduces ubiquitination of Lys676 suggesting that Lys676 is physiologically ubiquitinated by CRL4CRBN. The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro. This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.

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