E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines in vitro, knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix–loop–helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.

The usefulness of genome editing lies in the ability to induce any desirable change in the target genome. It is most efficiently achieved using ssDNA (sing

Objective To develop Deubiquitinase-Associated Signatures (DAS) to predict the prognosis of gynecological cancer patients. Method Using a cox-lasso regression model, we have developed Deubiquitinase-associated signatures for Cervical, Ovarian, and Uterine cancers. Developed DAS were validated in TCGA and GEO datasets. Survival analysis was carried out to know the effect of factors like menopausal stage and grade on DAS. The survival prediction accuracy of DAS was analyzed using ROC curves. Immune infiltration scores of 22 immune subtypes were explored using the CIBERSORT package in risk groups classified by DAS. Further, to target the unfavorable deubiquitinases (DUBs), compounds were identified using CMap database. Results Three DAS were developed for Cervical, Ovarian, and Uterine cancer types. DAS was able to predict survival and classify patients into two groups in TCGA and GEO datasets. DAS is an independent predictor of survival irrespective of tumor grade and menopausal stage. DAS, along with the clinical features, improves the accuracy of predictions. CIBERSORT analysis has shown that Immune cell infiltration is associated with risk groups divided by DAS. Using CMap, 52 compounds were identified to target unfavorable DUBs. Conclusion DAS is a good predictor of survival, and targeting unfavorable DUBs can decrease tumor progression in gynecological cancers. Synopsis Deubiquitinases (DUBs) are associated with cancer progression, limited studies on gynecological cancers. We used cox-lasso regression to develop a DUB-Associated prognostic Signature (DAS). DAS stratifies patients into high-low-risk and improves survival prediction accuracy. Small molecules were identified that can target poor prognostic DUBs.

DNA topoisomerase II beta (TOP2B) is crucial for the regulation of DNA topology, transcription, and genome stability. While its function has been extensively studied in post-mitotic cells such as neurons and cardiomyocytes, its role in proliferative cells, especially in the context of cellular senescence and aging, remains inadequately understood. Emerging evidence suggests that TOP2B expression declines with age and is implicated in several age-associated disorders, yet the molecular basis of its involvement in senescence remains poorly understood. In this investigation, we have specifically disrupted the promoter region of TOP2B in HEK 293T cells using CRISPR-Cas9 genome editing. The targeted region, spanning −533 to −481, which encompasses binding motifs for NF–Y and SP1, also accounts for a major proportion of TOP2B promoter activity. Knockout of this regulatory segment resulted in a marked reduction of TOP2B expression. The knockout cells exhibited classical features of senescence, including elevated ROS levels, increased senescence-associated β-galactosidase (SA-β-Gal) activity, and upregulation of key markers such as p53, p21, and p16.To examine the paracrine effects of senescence, conditioned media from TOP2B-deficient cells were given to wild-type HEK 293T cells, which also developed senescence-like phenotypes. These findings suggest that Loss of TOP2B expression is associated with the induction of senescence-like phenotypes in dividing cells and can propagate senescent signaling to neighboring cells. This study highlights a previously underappreciated role of TOP2B in the regulation of cellular aging and opens new avenues for understanding its potential contribution to age-related pathologies in proliferative cell types.