No abstract

No abstract

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild‐type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight‐binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30‐fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

No abstract

MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX.Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs – enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

AimsAmyloidosis is caused by deposition of abnormal protein fibrils, leading to damage of organ function. Hereditary amyloidosis represents a monogenic disease caused by germline mutations in 11 amyloidogenic precursor protein genes. One of the important but non-specific symptoms of amyloidosis is hypertrophic cardiomyopathy. Diagnostics of hereditary amyloidosis is complicated and the real cause can remain overlooked. We aimed to design hereditary amyloidosis gene panel and to introduce new next-generation sequencing (NGS) approach to investigate hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance.MethodsDesign of target enrichment DNA library preparation using Haloplex Custom Kit containing 11 amyloidogenic genes was followed by MiSeq Illumina sequencing and bioinformatics identification of germline variants using tool VarScan in a cohort of 40 patients.ResultsWe present design of NGS panel for 11 genes (TTR, FGA, APOA1, APOA2, LYZ, GSN, CST3, PRNP, APP, B2M, ITM2B) connected to various forms of amyloidosis. We detected one mutation, which is responsible for hereditary amyloidosis. Some other single nucleotide variants are so far undescribed or rare variants or represent common polymorphisms in European population.ConclusionsWe report one positive case of hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance and set up first panel for NGS in hereditary amyloidosis. This work may facilitate successful implementation of the NGS method by other researchers or clinicians and may improve the diagnostic process after validation.

Waldenström′s macroglobulinemia (WM) is a complex disease characterized by apparent morphological heterogeneity within the malignant clonal cells representing a continuum of small lymphocytes, plasmacytoid lymphocytes, and plasma cells. At the molecular level, the neoplastic B cell-derived clone has undergone somatic hypermutation, but not isotype switching, and retains the capability of plasmacytic differentia- | REGULATION OF B-CELL DEVELOPMENT: WHERE IS THE MISTAKE?Tumor cells of Waldenström′s macroglobulinemia derive from B lymphocyte during its development and show a wide morphological heterogeneity. 12 In general, the process of B lymphocyte development is regulated by several transcription factors and is accompa-