RESUMO
ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the misfolded proteins are dislocated from ER membrane into cytosol, which requires the AAA ATPase p97/VCP and its cofactor, the NPL4-UFD1 dimer. Here, we performed a CRISPR-based screen and identify many candidates for ERAD regulation. We further confirmed four proteins, FBOX2, TRIM6, UFL1 and WDR20, are novel regulators for ERAD. Then the molecular mechanism for WDR20 in ERAD is further characterized. Depletion of WDR20 inhibits the degradation of TCRα, a typical ERAD substrate, while WDR20 overexpression reduces TCRα protein level. WDR20 associates with TCRα and central regulators of the ERAD system, p97, GP78 and HRD1. A portion of WDR20 localizes to the ER-containing microsomal membrane. WDR20 expression increases TCRα ubiquitination, and HRD1 E3 ligase is essential for the process. WDR20 seems to serve as an adaptor protein to mediate the interaction between p97 and TCRα. Our study provides novel candidates and reveals an unexpected role of WDR20 in ERAD regulation.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Degradação Associada com o Retículo Endoplasmático , Adenosina Trifosfatases/metabolismo , Sistemas CRISPR-Cas , Proteínas de Transporte/química , Linhagem Celular Tumoral , Células HEK293 , Humanos , Microssomos/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , UbiquitinaçãoRESUMO
Previously, we reported that genomic loss of 14q occurs more frequently in high-grade than in low-grade clear cell renal cell carcinomas (ccRCCs), and has a significant impact on the levels of expression of genes located in this region, suggesting that such genes may be involved in the malignant transformation of ccRCCs. Here, we found that six of the genes located in the minimal common region of 14q loss were significantly downregulated in high-grade ccRCCs due to copy number loss. Using a dataset from The Cancer Genome Atlas Research Network, we found that downregulation of one of these six genes, WDR20, was significantly associated with poorer outcome in patients with ccRCC, suggesting that WDR20 downregulation may be involved in the malignant transformation of ccRCCs. In functional assays, exogenous WDR20 significantly inhibited the growth of RCC cell lines and induced apoptosis. Interestingly, the phosphorylation levels of ERK and protein kinase B/AKT, which reportedly contribute to the malignant phenotype of RCC cells, were clearly reduced by exogenous expression of WDR20. Thus, our data suggest that downregulation of WDR20 due to 14q loss may be involved in the malignant transformation of ccRCCs, in part through activation of the ERK and protein kinase B/AKT pathways.
Assuntos
Carcinoma de Células Renais/genética , Proteínas de Transporte/biossíntese , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Apoptose/genética , Carcinoma de Células Renais/patologia , Proteínas de Transporte/genética , Cromossomos Humanos Par 14 , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteína Oncogênica v-akt/genéticaRESUMO
The androgen receptor (AR), a member of the nuclear receptor family, is a transcription factor involved in prostate cell growth, homeostasis, and transformation. AR is a key protein in growth and development of both normal and malignant prostate, making it a common therapeutic target in prostate cancer. AR is regulated by an interplay of multiple post-translational modifications including ubiquitination. We and others have shown that the AR is ubiquitinated by a number of E3 ubiquitin ligases, including MDM2, CHIP, and NEDD4, which can result in its proteosomal degradation or enhanced transcriptional activity. As ubiquitination of AR causes a change in AR activity or stability and impacts both survival and growth of prostate cancer cells, deubiquitination of these sites has an equally important role. Hence, deubiquitinating enzymes could offer novel therapeutic targets. We performed an siRNA screen to identify deubiquitinating enzymes that regulate AR; in that screen ubiquitin-specific protease 12 (Usp12) was identified as a novel positive regulator of AR. Usp12 is a poorly characterized protein with few known functions and requires the interaction with two cofactors, Uaf-1 and WDR20, for its enzymatic activity. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, deubiquitinates the AR to enhance receptor stability and transcriptional activity. Our data show that Usp12 acts in a pro-proliferative manner by stabilizing AR and enhancing its cellular function.
Assuntos
Proliferação de Células , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Estabilidade Proteica , Receptores Androgênicos/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Despite its potential clinical relevance, the product of the DMWD (dystrophia myotonica, WD repeat containing) gene is a largely uncharacterized protein. The DMWD amino acid sequence is similar to that of WDR20, a known regulator of the USP12 and USP46 deubiquitinases (DUBs). Here, we apply a combination of in silico and experimental methods to investigate several aspects of DMWD biology. Molecular evolution and phylogenetic analyses reveal that WDR20 and DMWD, similar to USP12 and USP46, arose by duplication of a common ancestor during the whole genome duplication event in the vertebrate ancestor lineage. The analysis of public human gene expression datasets suggests that DMWD expression is positively correlated with USP12 expression in normal tissues and negatively correlated with WDR20 expression in tumors. Strikingly, a survey of the annotated interactome for DMWD and WDR20 reveals a largely nonoverlapping set of interactors for these proteins. Experimentally, we first confirmed that DMWD binds both USP12 and USP46 through direct coimmunoprecipitation of epitope-tagged proteins. We found that DMWD and WDR20 share the same binding interface in USP12, suggesting that their interaction with the DUB may be mutually exclusive. Finally, we show that both DMWD and WDR20 promote USP12 enzymatic activity, but they differentially modulate the subcellular localization of the DUB. Altogether, our findings suggest a model whereby mutually exclusive binding of DMWD and WDR20 to USP12 may lead to formation of deubiquitinase complexes with distinct subcellular localization, potentially targeting different substrate repertoires.
Assuntos
Proteínas de Transporte/metabolismo , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Distrofia Miotônica/patologia , Proteínas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Repetições WD40 , Sequência de Aminoácidos , Proteínas de Transporte/genética , Endopeptidases/genética , Evolução Molecular , Perfilação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Filogenia , Ligação Proteica , Proteínas/genética , Homologia de Sequência , Ubiquitina Tiolesterase/genéticaRESUMO
The human deubiquitinases USP12 and USP46 are very closely related paralogs with critical functions as tumor suppressors. The catalytic activity of these enzymes is regulated by two cofactors: UAF1 and WDR20. USP12 and USP46 show nearly 90% amino acid sequence identity and share some cellular activities, but have also evolved non-overlapping functions. We hypothesized that, correlating with their functional divergence, the subcellular localization of USP12 and USP46 might be differentially regulated by their cofactors. We used confocal and live microscopy analyses of epitope-tagged proteins to determine the effect of UAF1 and WDR20 on the localization of USP12 and USP46. We found that WDR20 differently modulated the localization of the DUBs, promoting recruitment of USP12, but not USP46, to the plasma membrane. Using site-directed mutagenesis, we generated a large set of USP12 and WDR20 mutants to characterize in detail the mechanisms and sequence determinants that modulate the subcellular localization of the USP12/UAF1/WDR20 complex. Our data suggest that the USP12/UAF1/WDR20 complex dynamically shuttles between the plasma membrane, cytoplasm and nucleus. This shuttling involved active nuclear export mediated by the CRM1 pathway, and required a short N-terminal motif (1MEIL4) in USP12, as well as a novel nuclear export sequence (450MDGAIASGVSKFATLSLHD468) in WDR20. In conclusion, USP12 and USP46 have evolved divergently in terms of cofactor binding-regulated subcellular localization. WDR20 plays a crucial role in as a "targeting subunit" that modulates CRM1-dependent shuttling of the USP12/UAF1/WDR20 complex between the plasma membrane, cytoplasm and nucleus.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Ubiquitina Tiolesterase/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sequência de Aminoácidos , Células HEK293 , Células HeLa , Humanos , Carioferinas/metabolismo , Modelos Biológicos , Sinais de Exportação Nuclear , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Ubiquitina Tiolesterase/química , Proteína Exportina 1RESUMO
Posttranslational modification of proteins by ubiquitin regulates synapse development and synaptic transmission. Much progress has been made investigating the role of ubiquitin ligases at the synapse, however very little is known about the deubiquitinating enzymes (DUBs) which remove ubiquitin from target proteins. Although there are far fewer DUBs than ubiquitin ligases encoded by the human genome, it is becoming clear that DUBs have very specific physiological functions, suggesting that DUB activity is tightly regulated in vivo. Many DUBs function as part of larger protein complexes, and multiple regulatory mechanisms exist to control the expression, localization and catalytic activity of DUBs. In this review article, we focus on the role of the DUB USP46 in the nervous system, and illustrate potential mechanisms of regulating DUBs by describing how USP46 is regulated by two WD40-repeat (WDR) proteins, WDR48/UAF1 and WDR20, based on recent structural studies and genetic analyses in vivo.