USP15, activated by TFAP4 transcriptionally, stabilizes SHC1 via deubiquitination and deteriorates renal cell carcinoma.

Ubiquitin-specific peptidase 15 (USP15), a critical deubiquitinating enzyme, has been demonstrated to improve substrate stabilization by hydrolyzing the bond between the substrate and ubiquitin, and is implicated in multiple carcinogenic processes. Prompted by the information cited from The Cancer Genome Atlas (TCGA) database and the Cancer Proteogenomic Data Analysis Site (cProSite), USP15 is selectively overexpressed in clear cell renal cell carcinoma (ccRCC) samples. We aimed to investigate the function of USP15 on ccRCC malignant features, which was emphasized in its deubiquitination of SHC adaptor protein 1 (SHC1). The overexpression of USP15 promoted the capacity of proliferation, migration, and invasion in ccRCC CAKI1 and 769-P cells, and these malignant biological properties were diminished by USP15 deletion in 786-O cells. USP15 accelerated tumor growth and lung metastasis in vivo. In addition, deubiquitinase USP15 was further identified as a new protector for SHC1 from degradation by the ubiquitination pathway, the post-translational modification. In sequence, transcription factor activating enhancer binding protein 4 (TFAP4) was shown to be partly responsible for USP15 expression at the level of transcription, as manifested by the chromatin immunoprecipitation and pull-down assay. Based on the in vitro and in vivo data, we postulate that USP15 regulated by TFAP4 transcriptionally deteriorates ccRCC malignant biological properties via stabilizing SHC1 by deubiquitination.

Identification and validation of a ferroptosis-related lncRNA signature to robustly predict the prognosis, immune microenvironment, and immunotherapy efficiency in patients with clear cell renal cell carcinoma.

Background: Ferroptosis is a new type of iron- and reactive oxygen species-dependent cell death, studies on ferroptosis-related long noncoding RNAs (FerLncRNAs) in clear cell renal cell carcinoma (ccRCC) are limited. The purpose of this study was to investigate the potential prognostic value of FerLncRNAs and their relationship with the immune microenvironment and immunotherapy response of ccRCC. Methods: RNA sequencing data of 526 patients with ccRCC were downloaded from The Cancer Genome Atlas (TCGA) database. The patients with ccRCC in TCGA were randomly divided (1:1) into a training and testing cohort. ICGC and GEO databases were used for validation. Screening for FerLncRNAs was performed using Pearson's correlation analysis with the reported ferroptosis-related genes. A FerLncRNA signature was constructed using univariate, LASSO, and multivariate Cox regression analyses in the training cohort. Internal and external datasets were performed to verify the FRlncRNA signature. Four major FRlncRNAs were verified through in vitro experiment. Results: We identified seven FerLncRNAs (LINC00894, DUXAP8, LINC01426, PVT1, PELATON, LINC02609, and MYG1-AS1), and established a risk signature and nomogram for predicting the prognosis of ccRCC. Four major FRlncRNAs were verified with the prognosis of ccRCC in the GEPIA and K-M Plotter databases, and their expressions were validated by realtime PCR. The risk signature can also effectively reflect the immune environment, immunotherapy response and drug sensitivity of ccRCC. These FRlncRNAs have great significance to the implementation of individualized treatment and disease monitoring of ccRCC patients.

An integrated biophysical approach to discovering mechanisms of NDM-1 inhibition for several thiol-containing drugs.

Due to the rapid proliferation of antibiotic-resistant pathogenic bacteria, known as carbapenem-resistant enterobacteriaceae, the efficacy of ß-lactam antibiotics is threatened. ß-lactam antibiotics constitute over 50% of the available antibiotic arsenal. Recent efforts have been focused on developing inhibitors to these enzymes. In an effort to understand the mechanism of inhibition(s) of four FDA-approved thiol-containing drugs that were previously reported to be inhibitors of New Delhi metallo-ß-lactamase (NDM-1), various biochemical and spectroscopic techniques were used. Isothermal titration calorimetry demonstrated the binding affinity to NDM-1 corresponds to the reported IC50 values of the inhibitors. Equilibrium dialyses and metal analyses demonstrated that all of these inhibitors formed ternary complexes with ZnZn-NDM-1. Spectroscopic studies on CoCo-NDM-1 revealed two distinct binding modes for the thiol-containing compounds. These findings validate the need to further investigate the mechanism of inhibition of MBL inhibitors. Further research to identify inhibition capabilities beyond reported IC50 values is necessary for understanding the binding modes of these identified compounds and to provide the necessary foundation for developing clinically relevant MBL inhibitors.

δ-Catenin regulates proliferation and apoptosis in renal cell carcinoma via promoting ß-catenin nuclear localization and activating its downstream target genes.

δ-Catenin is a unique member of the catenin family and is proved to be overexpressed in diverse human cancer types. However, the clinical significance and underling mechanism of δ-catenin expression in renal cell carcinoma (RCC) remain elusive. Herein, we detected the protein expression of δ-catenin in 28 clinical specimens of paired renal cancer tissues and normal renal tissues by Western blot analysis. δ-Catenin expression in 58 cases of renal cell carcinoma was also examined by immunohistochemistry, and its association with clinicopathological factors was analyzed by statistical analysis. In vitro and in vivo assays were employed to further explore the biological role of δ-catenin in RCC. The results showed that δ-catenin was highly expressed in both clinical samples and cell lines of RCC. RCC patients with higher δ-catenin expression had a more advanced pTNM stage and tumor stage as well as lymph nodes metastasis than those with lower expression. By regulating the nuclear translocation of ß-catenin and ß-catenin-mediated oncogenic signals, δ-catenin promoted proliferation and inhibited apoptosis in RCC. In vivo assay indicated δ-catenin facilitated tumor growth in ACHN cell xenograft mouse model. Taken together, our study suggests that δ-catenin might be considered as a novel prognostic indicator and actionable target for gene therapy in renal cell carcinoma.

Evolution of New Delhi metallo-ß-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used ß-lactamase inhibitors to counter the activity of metallo-ß-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-ß-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of ß-lactam antibiotics and zinc deprivation.

The Continuing Challenge of Metallo-ß-Lactamase Inhibition: Mechanism Matters.

Metallo-ß-lactamases (MBLs) are a significant clinical problem because they hydrolyze and inactivate nearly all ß-lactam-containing antibiotics. These 'lifesaving drugs' constitute >50% of the available contemporary antibiotic arsenal. Despite the global spread of MBLs, MBL inhibitors have not yet appeared in clinical trials. Most MBL inhibitors target active site zinc ions and vary in mechanism from ternary complex formation to metal ion stripping. Importantly, differences in mechanism can impact pharmacology in terms of reversibility, target selectivity, and structure-activity relationship interpretation. This review surveys the mechanisms of MBL inhibitors and describes methods that determine the mechanism of inhibition to guide development of future therapeutics.