Dataset Information

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells.

ABSTRACT: Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells in vitro and in vivo However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor suppressor targets of E6AP, promyelocytic leukemia protein and p27. To explore additional players that are regulated downstream of E6AP, we combined a transcriptomic and proteomic approach. We identified and quantified 16,130 transcripts and 7,209 proteins in castration resistant prostate cancer cell line, DU145. A total of 2,763 transcripts and 308 proteins were significantly altered on knockdown of E6AP. Pathway analyses supported the known phenotypic effects of E6AP knockdown in prostate cancer cells and in parallel exposed novel potential links of E6AP with cancer metabolism, DNA damage repair and immune response. Changes in expression of the top candidates were confirmed using real-time polymerase chain reaction. Of these, clusterin, a stress-induced chaperone protein, commonly deregulated in prostate cancer, was pursued further. Knockdown of E6AP resulted in increased clusterin transcript and protein levels in vitro and in vivo Concomitant knockdown of E6AP and clusterin supported the contribution of clusterin to the phenotype induced by E6AP. Overall, results from this study provide insight into the potential biological pathways controlled by E6AP in prostate cancer cells and identifies clusterin as a novel target of E6AP.

SUBMITTER: Gulati T

PROVIDER: S-EPMC5986238 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells.

Gulati Twishi T Huang Cheng C Caramia Franco F Raghu Dinesh D Paul Piotr J PJ Goode Robert J A RJA Keam Simon P SP Williams Scott G SG Haupt Sue S Kleifeld Oded O Schittenhelm Ralf B RB Gamell Cristina C Haupt Ygal Y

Molecular & cellular proteomics : MCP 20180220 6

Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells <i>in vitro</i> and <i>in vivo</i> However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor su ...[more]

PMID: 29463595

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Project description:The degradation of p53 is a hallmark of high-risk human papillomaviruses (HPVs) of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely related HPV16 and HPV31. HPV16 E6 and HPV31 E6 display high sequence identity (∼67%). We report here, for the first time, the structure of HPV31 E6 bound to the LxxLL motif of E6AP. HPV16 E6 and HPV31 E6 are structurally very similar, in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31 E6 to the LxxLL motif of E6AP and p53, respectively, are reduced 2-fold and 5.4-fold compared to 16 E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely, degradation of p53. Therefore, closely related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathological impacts.IMPORTANCE Variations of carcinogenicity of human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely related HPVs, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related high-risk HPV31 and HPV16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31 E6 bound to the E6AP LxxLL motif. HPV16 E6 and 31 E6 structures are highly similar, but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31 E6 than 16 E6. These results align with the observed lower p53 degradation potential of 31 E6.

Dataset Information

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells.

Publications

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells.

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