Structural and functional characterization of USP47 reveals a hot spot for inhibitor design.

USP47 is widely involved in tumor development, metastasis, and other processes while performing a more regulatory role in inflammatory responses, myocardial infarction, and neuronal development. In this study, we investigate the functional and biochemical properties of USP47, whereby depleting USP47 inhibited cancer cell growth in a p53-dependent manner-a phenomenon that enhances during the simultaneous knockdown of USP7. Full-length USP47 shows higher deubiquitinase activity than the catalytic domain. The crystal structures of the catalytic domain, in its free and ubiquitin-bound states, reveal that the misaligned catalytic triads, ultimately, become aligned upon ubiquitin-binding, similar to USP7, thereby becoming ready for catalysis. Yet, the composition and lengths of BL1, BL2, and BL3 of USP47 differ from those for USP7, and they contribute to the observed selectivity. Our study provides molecular details of USP47 regulation, substrate recognition, and the hotspots for drug discovery by targeting USP47.

Safety and Effectiveness of Empagliflozin in Korean Patients with Type 2 Diabetes Mellitus: Results from a Nationwide Post-Marketing Surveillance.

BACKGROUND: To evaluate the safety and effectiveness of empagliflozin in routine clinical settings, we collected and assessed the clinical profiles of Korean patients with type 2 diabetes mellitus. METHODS: This was a post-marketing surveillance study of empagliflozin 10 and 25 mg. Information on adverse events and adverse drug reactions (ADRs) was collected as safety data sets. Available effectiveness outcomes, including glycosylated hemoglobin (HbA1c) level, fasting plasma glucose, body weight, and blood pressure, were assessed. RESULTS: The incidence rate of ADRs was 5.14% in the safety dataset (n=3,231). Pollakiuria, pruritis genital, and weight loss were the most common ADRs. ADRs of special interest accounted for only 1.18%, and there were no serious events that led to mortality or hospitalization. In the effectiveness data set (n=2,567), empagliflozin significantly reduced the mean HbA1c level and body weight during the study period by -0.68%±1.39% and -1.91±3.37 kg (both P<0.0001), respectively. In addition, shorter disease duration, absence of dyslipidemia, and higher baseline HbA1c levels were identified as the clinical features characteristic of a "responder" to empagliflozin therapy. CONCLUSION: Empagliflozin is a safe and potent glucose-lowering drug in routine use among Korean patients with type 2 diabetes mellitus. It is expected to have better glycemic efficacy in Korean patients with poorly controlled type 2 diabetes mellitus.

MicroRNA-101-3p Suppresses Cancer Cell Growth by Inhibiting the USP47-Induced Deubiquitination of RPL11.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate a countless number of genes in the cell, and the aberrant expression of miRNA can lead to cancer. Here, we demonstrate that miR-101-3p regulates the RPL11-MDM2-p53 pathway by targeting ubiquitin-specific peptidase 47 (USP47), consequently inhibiting cancer cell proliferation. We confirm that miR-101-3p directly binds to the 3'-UTR region of the USP47 gene and inhibits USP47 expression. In addition, the overexpression of miR-101-3p suppresses cell proliferation in a p53-dependent manner. MiR-101-3p promotes interaction between RPL11 and MDM2 by inducing the translocation of RPL11 from the nucleolus to the nucleoplasm, thus preventing the MDM2-mediated proteasomal degradation of p53. However, these phenomena are restored by the overexpression of USP47, but not by its catalytically inactive form. Indeed, miR-101-3p regulates RPL11 localization and its interaction with MDM2 by inhibiting the USP47-induced deubiquitination of RPL11. Finally, the expression of miR-101-3p is downregulated in lung cancer patients, and the patients with low miR-101-3p expression exhibit a lower survival rate, indicating that miR-101-3p is associated with tumorigenesis. Together, our findings suggest that miR-101-3p functions as a tumor suppressor by targeting USP47 and could be a potential therapeutic target for cancers.

Ubiquitin-proteasome system (UPS) as a target for anticancer treatment.

The ubiquitin-proteasome system (UPS) plays an important role in the cellular processes for protein quality control and homeostasis. Dysregulation of the UPS has been implicated in numerous diseases, including cancer. Indeed, components of UPS are frequently mutated or abnormally expressed in various cancers. Since Bortezomib, a proteasome inhibitor, received FDA approval for the treatment of multiple myeloma and mantle cell lymphoma, increasing numbers of researchers have been seeking drugs targeting the UPS as a cancer therapeutic strategy. Here, we introduce the essential component of UPS, including ubiquitinating enzymes, deubiquitinating enzymes and 26S proteasome, and we summarize their targets and mechanisms that are crucial for tumorigenesis. In addition, we briefly discuss some UPS inhibitors, which are currently in clinical trials as cancer therapeutics.

Assay Systems for Profiling Deubiquitinating Activity.

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein-protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

USP47 Promotes Tumorigenesis by Negative Regulation of p53 through Deubiquitinating Ribosomal Protein S2.

p53 is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. Although the regulations of p53 by E3 ligase and deubiquitinating enzymes (DUBs) have been described, the cellular roles of DUB associated with ribosomal stress have not been well studied. In this study, we report that Ubiquitin Specific Protease 47 (USP47) functions as an important regulator of p53. We show that ubiquitinated ribosomal protein S2 (RPS2) by Mouse double minute 2 homolog (MDM2) is deubiquitinated by USP47. USP47 inhibits the interaction between RPS2 and MDM2 thereby alleviating RPS2-mediated suppression of MDM2 under normal conditions. However, dissociation of USP47 leads to RPS2 binding to MDM2, which is required for the suppression of MDM2, consequently inducing up-regulation of the p53 level under ribosomal stress. Finally, we show that depletion of USP47 induces p53 and therefore inhibits cell proliferation, colony formation, and tumor progression in cancer cell lines and a mouse xenograft model. These findings suggest that USP47 could be a potential therapeutic target for cancer.

Ribosomal protein S2 interplays with MDM2 to induce p53.

The MDM2-p53 pathway is crucial for maintenance of p53 homeostasis. Some ribosomal proteins (RPs) play critical roles in regulating p53 by interacting with MDM2. However, the role and functional mechanism of each RP in MDM2-p53 pathway still remain unknown. In this study, we found that Ribosomal Protein S2 (RPS2) is a new regulator of MDM2-P53 signaling pathway to regulate p53 protein level. Here, we characterized that RPS2 interacts with MDM2 through the RING finger domain of MDM2. RPS2 is ubiquitinated by MDM2 and the ubiquitinated status of RPS2 regulates the stability of p53, which is activated in response to cellular stresses such as DNA damage, oxidative stress, and especially ribosomal stress. In addition, p53 is not induced in RPS2 knockdown even in the ribosomal stressed condition, indicating that RPS2 is essential for the stabilization of p53. Collectively, our data suggest that RPS2 plays a critical role in the regulation of p53 signaling including the ribosomal stress response.

Deubiquitinating Enzymes: A Critical Regulator of Mitosis.

Mitosis is a complex and dynamic process that is tightly regulated by a large number of mitotic proteins. Dysregulation of these proteins can generate daughter cells that exhibit genomic instability and aneuploidy, and such cells can transform into tumorigenic cells. Thus, it is important for faithful mitotic progression to regulate mitotic proteins at specific locations in the cells at a given time in each phase of mitosis. Ubiquitin-dependent modifications play critical roles in this process by regulating the degradation, translocation, or signal transduction of mitotic proteins. Here, we review how ubiquitination and deubiquitination regulate the progression of mitosis. In addition, we summarize the substrates and roles of some deubiquitinating enzymes (DUBs) crucial for mitosis and describe how they contribute error correction during mitosis and control the transition between the mitotic phases.

Structural basis for ovarian tumor domain-containing protein 1 (OTU1) binding to p97/valosin-containing protein (VCP).

Valosin-containing protein (VCP), also known as p97, is an AAA(+) ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXLOTU1) complexed to the N-terminal domain of VCP (NVCP) at 1.8-Å resolution reveals that UBXLOTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of NVCP using the (39)GYPP(42) loop of UBXLOTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a KD of 0.71 µM. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.