HPV E6, E6AP and cervical cancer.

Every year, approximately 470,000 new cases of cervical cancer are diagnosed and approximately 230,000 women worldwide die of the disease, with the majority (approximately 80%) of these cases and deaths occurring in developing countries. Human papillomaviruses (HPVs) are the etiological agents in nearly all cases (99.7%) of cervical cancer, and the HPV E6 protein is one of two viral oncoproteins that is expressed in virtually all HPV-positive cancers. E6 hijacks a cellular ubiquitin ligase, E6AP, resulting in the ubiquitylation and degradation of the p53 tumor suppressor, as well as several other cellular proteins. While the recent introduction of prophylactic vaccines against specific HPV types offers great promise for prevention of cervical cancer, there remains a need for therapeutics. Biochemical characterization of E6 and E6AP has suggested approaches for interfering with the activities of these proteins that could be useful for this purpose. PUBLICATION HISTORY : Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

Human papillomavirus E6-induced degradation of E6TP1 is mediated by E6AP ubiquitin ligase.

High-risk human papilloma viruses are known to be associated with cervical cancers. We have reported previously that the high-risk human papillomavirus (HPV) E6 oncoprotein interacts with E6TP1, a novel Rap GTPase-activating protein (RapGAP). Similar to p53 tumor suppressor protein, the high-risk HPV E6 oncoproteins target E6TP1 for degradation. The HPV16 E6-induced degradation of E6TP1 strongly correlates with its ability to immortalize human mammary epithelial cells. In this study, we used treatment with a proteasome inhibitor MG132, analysis in CHO-ts20 cells with a thermolabile ubiquitin-activating enzyme, and direct detection of ubiquitin-modified E6TP1 to demonstrate that E6TP1 is targeted for degradation by the ubiquitin-proteasome pathway both in the presence and in the absence of E6. Using deletion mutants of E6TP1, we mapped the region required and sufficient for E6 binding to COOH-terminal 40 amino acid residues and showed this region to be necessary for E6-dependent degradation of E6TP1. Furthermore, the E6-binding region of E6TP1 complexes with E6AP via E6. Importantly, the purified E6AP enhanced the ubiquitination and degradation of E6TP1 in the presence of E6 in vitro. Additionally, the expression of a dominant-negative E6AP mutant (C833A) in cells inhibited the E6-induced degradation of E6TP1. These findings demonstrate that the E6-induced decrease in the levels of E6TP1 protein involves the E6AP-mediated ubiquitination followed by proteasome-dependent degradation.

High-level expression and purification of recombinant E1 enzyme.

The ubiquitin E1 enzyme is an ATP-dependent enzyme that activates ubiquitin for use in all ubiquitin conjugation pathways. This chapter describes the expression and purification of human E1 enzyme for use in in vitro ubiquitination reactions.

Expression and assay of HECT domain ligases.

HECT domain ubiquitin ligases (HECT E3s), typified by human E6AP and yeast Rsp5p, are unique among the several classes of known ubiquitin ligases in that they participate directly in the chemistry of substrate ubiquitination reactions. This chapter discusses strategies for the expression of active HECT E3s and the assays that are available for analyzing E2 interaction, ubiquitin-thioester formation, and substrate ubiquitination.

Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel.

BACKGROUND: Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures. METHODS: The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance. RESULTS: We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10(-16)), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CI = 56.3, 92.5) with 100% PPV (95% CI = 81.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CI = 66.4,97.2) while maintaining PPV. CONCLUSIONS: The results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens.

Targeted, high-depth, next-generation sequencing of cancer genes in formalin-fixed, paraffin-embedded and fine-needle aspiration tumor specimens.

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.

Herc5, an interferon-induced HECT E3 enzyme, is required for conjugation of ISG15 in human cells.

ISG15 is an interferon (IFN)-alpha/beta-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections. A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-beta, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.

The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-alpha/beta-induced ubiquitin-like protein.

Ubiquitin-(Ub) like proteins (Ubls) are conjugated to their targets by an enzymatic cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and in some cases an E3 ligase. ISG15 is a Ubl that is conjugated to cellular proteins after IFN-alpha/beta stimulation. Although the E1 enzyme for ISG15 (Ube1L/E1(ISG15)) has been identified, the identities of the downstream components of the ISG15 conjugation cascade have remained elusive. Here we report the purification of an E2 enzyme for ISG15 and demonstrate that it is UbcH8, an E2 that also functions in Ub conjugation. In vitro assays with purified Ub E2 enzymes and in vivo RNA interference assays indicate that UbcH8 is a major E2 enzyme for ISG15 conjugation. These results indicate that the ISG15 conjugation pathway overlaps or converges with the Ub conjugation pathway at the level of a specific E2 enzyme. Furthermore, these results raise the possibility that the ISG15 conjugation pathway might use UbcH8-competent Ub ligases in vivo. As an initial test of this hypothesis, we have shown that a UbcH8-competent Ub ligase conjugates ISG15 to a specific target in vitro. These results challenge the concept that Ub and Ubl conjugation pathways are strictly parallel and nonoverlapping and have important implications for understanding the regulation and function of ISG15 conjugation in the IFN-alpha/beta response.