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The deubiquitylase USP7 is a novel cyclin F-interacting protein and regulates cyclin F protein stability.

ABSTRACT: Cyclin F, unlike canonical and transcriptional cyclins, does not bind or activate any cyclin-dependent kinases. Instead, it harbors an F-box motif and primarily functions as the substrate recognition subunit of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF^Cyclin ^F. By targeting specific proteins for ubiquitin-mediated proteasomal degradation, cyclin F plays a critical role in the regulation of centrosomal duplication, DNA replication and repair, and maintenance of genomic stability. Cyclin F abundance and activity are tightly regulated throughout the cell cycle. However, the molecular mechanisms regulating cyclin F are scantily understood. Here, we identify the deubiquitylase USP7 as a novel cyclin F-interacting protein. We observe that USP7 stabilizes cyclin F protein and that this function is independent of the deubiquitylase activity of USP7. Additionally, our data suggest that USP7 is also involved in the regulation of cyclin F mRNA. Pharmacological inhibition of the deubiquitylase activity of USP7 resulted in downregulation of cyclin F mRNA.

SUBMITTER: Sharma SS

PROVIDER: S-EPMC9699750 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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The deubiquitylase USP7 is a novel cyclin F-interacting protein and regulates cyclin F protein stability.

Sharma Savitha S SS Pledger W Jack WJ Kondaiah Paturu P

Aging 20221105 21

Cyclin F, unlike canonical and transcriptional cyclins, does not bind or activate any cyclin-dependent kinases. Instead, it harbors an F-box motif and primarily functions as the substrate recognition subunit of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF<sup>Cyclin</sup> <sup>F</sup>. By targeting specific proteins for ubiquitin-mediated proteasomal degradation, cyclin F plays a critical role in the regulation of centrosomal duplication, DNA replication and repair, and maintenance of ge ...[more]

PMID: 36342772

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Project description:BackgroundAccumulating evidence has demonstrated that aberrant expression of deubiquitinating enzymes is associated with the initiation and progression of Triple-negative breast cancer (TNBC). The publicly available TCGA database of breast cancer data was used to analyze the OTUD deubiquitinating family members that were correlated with survival of breast cancer and ovarian tumor domain-containing 2 (OTUD-2), or YOD1 was identified. The aim of present study was to assess YOD1 expression and function in human TNBC and then explored the underlying molecular events.MethodsWe detected the expression of YOD1 in 32 TNBC and 44 NTNBC samples by qRT-PCR, Western blot and immunohistochemistry. Manipulation of YOD1 expression was assessed in vitro and in vivo for TNBC cell proliferation, migration, invasion, cell-cycle and drug resistance, using colony formation assay, transwell assay, CCK8 assay, TUNEL assay, flow cytometric analysis and xenograft tumor assay. Next, proteomic analysis, Western blot, proximity ligation assay, Immunoprecipitation, and Immunofluorescence were conducted to assess downstream targets.ResultsIt was found that YOD1 was significantly upregulated in TNBC tissues compared with non-triple-negative breast cancer (NTNBC), which was positively correlated with poor survival in TNBC patients. Knockdown of YOD1 effectively inhibited TNBC cell migration, proliferation, cell cycle and resistance to cisplatin and paclitaxel. Mechanistically, YOD1 promoted TNBC progression in a manner dependent on its catalytic activity through binding with CDK1, leading to de-polyubiquitylation of CDK1 and upregulation of CDK1 expression. In addition, YOD1 overexpression was found to be correlated with CDK1 overexpression in human TNBC specimens. Finally, in vivo study demonstrated that YOD1 knockdown or YOD1 inhibitor could inhibit CDK1 expression and suppress the growth and metastasis of TNBC tumors.ConclusionOur study highlights that YOD1 functions as an oncogene in TNBC via binding to CDK1 and mediated its stability and oncogenic activity. Interfering with YOD1 expression or YOD1 inhibitor could suppress TNBC cells in vitro and in vivo, suggesting that YOD1 may prove to be a promising therapeutic target for TNBC.

Project description:BackgroundThe deubiquitinase USP7 has been identified as an oncogene with key roles in tumorigenesis and therapeutic resistance for a series of cancer types. Recently small molecular inhibitors have been developed to target USP7. However, the anticancer mechanism of USP7 inhibitors is still elusive.MethodsCell viability or clonogenicity was tested by violet crystal assay. Cell apoptosis or cell cycle was analyzed by flow cytometry, and chromosome misalignment was observed by a fluorescent microscopy. The protein interaction of PLK1 and USP7 was detected by tandem affinity purification and high throughput proteomics, and further confirmed by co-immunoprecipitation, GST pull-down and protein co-localization. The correlation between USP7 level of tumor tissues and taxane-resistance was evaluated.ResultsPharmacological USP7 inhibition by P5091 retarded cell proliferation and induced cell apoptosis. Further studies showed that P5091 induced cell cycle arrest at G2/M phase, and particularly induced chromosome misalignment, indicating the key roles of USP7 in mitosis. USP7 protein was detected in the PLK1-interacted protein complex. USP7 interacts with PLK1 protein through its PBD domain by catalytic activity. USP7 as a deubiquitinase sustained PLK1 protein stability via the C223 site, and inversely, USP7 inhibition by P5091 promoted the protein degradation of PLK1 through the ubiquitination-proteasome pathway. By overexpressing PLK1, USP7 that had been depleted by RNAi ceased to induce chromosome misalignment in mitosis and again supported cell proliferation and cell survival. Both USP7 and PLK1 were overexpressed in taxane-resistant cancer cells, and negatively correlated with the MP scores in tumor tissues. Either USP7 or PLK1 knockdown by RNAi significantly sensitized taxane-resistant cells to taxane cell killing.ConclusionThis is the first report that PLK1 is a novel substrate of USP7 deubiquitinase, and that USP7 sustained the protein stability of PLK1. USP7 inhibition induces cell apoptosis and cell cycle G2/M arrest, and overcomes taxane resistance by inducing the protein degradation of PLK1, resulting in chromosome misalignment in mitosis.

Dataset Information

The deubiquitylase USP7 is a novel cyclin F-interacting protein and regulates cyclin F protein stability.

Publications

The deubiquitylase USP7 is a novel cyclin F-interacting protein and regulates cyclin F protein stability.

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