Loss of Deubiquitinase USP1 Blocks Pancreatic β-Cell Apoptosis by Inhibiting DNA Damage Response.
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ABSTRACT:
SUBMITTER: Gorrepati KDD
PROVIDER: S-EPMC6135944 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Loss of insulin-secreting β-cells in diabetes may be either due to apoptosis or dedifferentiation of β-cell mass. The ubiquitin-proteasome system comprising E3 ligase and deubiquitinases (DUBs) controls several aspects of β-cell functions. In this study, screening for key DUBs identified USP1 to be specifically involved in dedifferentiation process. Inhibition of USP1 either by genetic intervention or small-molecule inhibitor ML323 restored epithelial phenotype of β-cells, but not with inhibition of other DUBs. In absence of dedifferentiation cues, overexpression of USP1 was sufficient to induce dedifferentiation in β-cells; mechanistic insight showed USP1 to mediate its effect via modulating the expression of inhibitor of differentiation (ID) 2. In an in vivo streptozotocin (STZ)-induced dedifferentiation mouse model system, administering ML323 alleviated hyperglycemic state. Overall, this study identifies USP1 to be involved in dedifferentiation of β-cells and its inhibition may have a therapeutic application of reducing β-cell loss during diabetes.
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Project description:SARS-CoV-2 belongs to the subgenus Sarbecovirus, which universally encodes the accessory protein ORF6. SARS-CoV-2 ORF6 is an antagonist of the interferon (IFN)-mediated antiviral response and plays an important role in viral infections. However, the mechanism by which the host counteracts the function of ORF6 to restrict viral replication remains unclear. In this study, we found that most ORF6 proteins encoded by sarbecoviruses could be ubiquitinated and subsequently degraded via the proteasome pathway. Through extensive screening, we identified that the deubiquitinase USP1, which effectively and broadly deubiquitinates sarbecovirus ORF6 proteins, stabilizes ORF6 proteins, resulting in enhanced viral replication. Therefore, ubiquitination and deubiquitination of ORF6 are important for antagonizing IFN-mediated antiviral signaling and influencing the virulence of SARS-CoV-2. These findings highlight an essential molecular mechanism and may provide a novel target for therapeutic interventions against viral infections.IMPORTANCEThe ORF6 proteins encoded by sarbecoviruses are essential for effective viral replication and infection and are important targets for developing effective intervention strategies. In this study, we confirmed that sarbecovirus ORF6 proteins are important antagonists of the host immune response and identified the regulatory mechanisms of ubiquitination and deubiquitination of most sarbecovirus ORF6 proteins. Moreover, we revealed that DUB USP1 prevents the proteasomal degradation of all ORF6 proteins, thereby promoting the virulence of SARS-CoV-2. Thus, impeding ORF6 function is helpful for attenuating the virulence of sarbecoviruses. Therefore, our findings provide a deeper understanding of the molecular mechanisms underlying sarbecovirus infections and offer potential new therapeutic targets for the prevention and treatment of these infections.
| S-EPMC10804995 | biostudies-literature
Project description:BackgroundOur previous study showed that the ribosomal protein L21 (RPL21) may play an important role in the development and survival of pancreatic cancer. In this article, RNA interference (RNAi) experiments were performed with RPL21-specific small interfering RNA (siRNA) to elucidate the mechanism by which RPL21 controls PC PANC-1 and BxPC-3 cell proliferation.MethodsIn the present study, PANC-1, BxPC-3 cells, and BALB/c nude mice were used to investigate antitumor effect and mechanism by which RPL21 controls cell proliferation and apoptosis in vitro and in vivo. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation, cell cycle and cell apoptosis in vitro were determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays and flow cytometry assay. The mechanism of RPL21 regulating cell proliferation was investigated using transcriptome sequencing analysis and luciferase reporter assay. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation in vivo were determined using BALB/c nude mice tumor model.ResultsIn PANC-1 and BxPC-3 cells, the knockdown of RPL21 expression with corresponding siRNA suppressed cell proliferation in vitro and in vivo, inhibited DNA replication, and induced arrests in the G1 phase of the cell cycle. Further results showed that the mini-chromosome maintenance (MCM) protein family (MCM2-7), CCND1 and CCNE1 were down-regulated significantly in PANC-1 and BxPC-3 cells after transfected with RPL21 siRNA, which suggests that the suppression of DNA replication is due to the reduced expression of MCM2-7 family, and the induction of G1 arrest is correlated with the inhibition of CCND1 and CCNE1. Luciferase reporter assay showed that RPL21 controls the DNA replication and G1-S phase progression possibly through the regulation of E2F1 transcription factor in PC cells. Moreover, RPL21 siRNA showed an apoptosis-inducing effect only in BxPC-3 and PANC-1 cells but not in normal HPDE6-C7 cells. The increase of caspase-8 activities and the loss of mitochondrial membrane potential after RPL21 silencing indicates that the RPL21 gene may be involved in caspase-8-related mitochondrial apoptosis.ConclusionOur findings suggest that siRNA against the RPL21 gene possesses a potential anti-cancer activity for PC cells by inhibiting their proliferation and DNA replication, as well as inducing cell cycle G1 arrest and cell apoptosis.
| S-EPMC7509406 | biostudies-literature
Project description:One of the major risk factors for asthma development is exposure to environmental allergens. House dust mites (HDM) can induce DNA damage, resulting in asthma. Resveratrol (RES) produced by several plants, has anti‑apoptotic properties and may affect a variety of biological processes. The aim of the present study was to investigate the protective role of RES against apoptosis in bronchial epithelial cells. C57BL/6J mice treated with HDM exhibited high levels of cell apoptosis, while RES significantly reversed this process. Induced DNA damage was more severe in the HDM group vs. the HDM combined with RES group. This result was confirmed by immunostaining and western blot analysis of the protein expression of the DNA damage‑related gene γH2AX, which was highly induced by HDM. In addition, treatment with RES protected bronchial epithelial cells exposed to HDM from DNA damage. RES decreases reactive oxygen species levels to inhibit oxidative DNA damage in bronchial epithelial cells. Furthermore, compared with the HDM group, induced cell apoptosis could be attenuated by RES in the group of combined treatment with RES and HDM. A DNA repair inhibitor augmented DNA damage and apoptosis in bronchial epithelial cells, whereas RES significantly attenuated cell apoptosis through inhibiting DNA damage.
| S-EPMC7169938 | biostudies-literature
Project description:Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.
| S-EPMC4144829 | biostudies-literature
Project description:Optimal activation of TANK-binding kinase 1 (TBK1) is crucial for initiation of innate antiviral immunity and maintenance of immune homeostasis. Although several E3 ubiquitin ligases have been reported to regulate TBK1 activation by mediating its polyubiquitination, the functions of deubiquitinase on TBK1 activity remain largely unclear. Here, we identified a deubiquitinase complex, which is formed by ubiquitin specific peptidase 1 (USP1) and USP1-associated factor 1 (UAF1), as a viral infection-induced physiological enhancer of TBK1 expression. USP1-UAF1 complex enhanced TLR3/4 and RIG-I-induced IFN regulatory factor 3 (IRF3) activation and subsequent IFN-? secretion. Mechanistically, USP1 and UAF1 bound to TBK1, removed its K48-linked polyubiquitination, and then reversed the degradation process of TBK1. Furthermore, we found that ML323, a specific USP1-UAF1 inhibitor, attenuated IFN-? expression and enhanced viral replication both in vitro and in vivo. Therefore, our results outline a novel mechanism for the control of TBK1 activity and suggest USP1-UAF1 complex as a potential target for the prevention of viral diseases.
| S-EPMC5716033 | biostudies-literature
Project description:The innate immune response against Helicobacter pylori is mainly controlled by pattern recognition receptors that lead to the up-regulation of pro-inflammatory cytokines. A new player in this field is the miR-155 being regulated by TLR ligands in monocytic derived cells influencing certain intracellular pathways by down-regulating targets involved in signaling and development. By using primary bone marrow derived macrophages (BMMs) from mice deficient in the key TLR signals the regulation of miR-155 was explored. Further on the biological impact of a lack of miR-155 in BMMs was analyzed by micro-array studies and apoptosis assays. We observed that miR-155 is up-regulated in response to H. pylori via TLR2 and TLR4 in a NF-M-NM-:B dependent manner, but also identified a TLR2/TLR4 independent mechanism that was strongly connected to a functional TypeIV secretion system. Down-stream the high expression of miR-155 down-regulated many mRNA targets during H. pylori infection. Thereby we could validate Tspan14, Lpin1, and Pmaip1 as new targets of miR-155 and identified their binding site. These and other already published targets showed a substantial pro-apoptotic potential. We observed that H. pylori infected wild type BMMs were much more resistant to DNA damage induced apoptosis by cisplatin when compared to their respective miR-155-/- BMMs. Our data strongly suggests that there is an additional innate immune mechanism of BMMs leading to the upregulation of the anti-apoptotic miR-155 that causes resistance to DNA damage in BMMs. Microarray experiments were performed as dual-color hybridizations. To compensate for dye-specific effects, an independent dye-reversal color-swap was applied. Bone marrow derived macrophages were 'in vitro' infected with H.pylori P12 and the expression changes were measured.
2012-04-02 | E-GEOD-29388 | biostudies-arrayexpress
Project description:The maintenance of genetic stability depends on the fine-tuned initiation and termination of pathways involved in cell cycle checkpoints and DNA repair. Here, we describe a new pathway that regulates checkpoint kinase 1 (CHK1) activity, a key element controlling both checkpoints and DNA repair. We show that the ubiquitin-specific peptidase 1 (USP1) deubiquitinase participates in the maintenance of both total and phosphorylated levels of CHK1 in response to genotoxic stress. We establish that USP1 depletion stimulates the damage-specific DNA-binding protein 1-dependent degradation of phosphorylated CHK1 in both a monoubiquitinylated Fanconi anaemia, complementation group D2 (FANCD2)-dependent and -independent manner. Our data support the existence of a circuit in which CHK1 activates checkpoints, DNA repair and proliferating cell nuclear antigen and FANCD2 monoubiquitinylation. The latter two events, in turn, switch off activated CHK1 by negative feedback inhibition, which contributes to the downregulation of the DNA damage response. This pathway, which is compromised in the cancer-prone disease Fanconi anaemia (FA), likely contributes to the hypersensitivity of cells from FA patients to DNA damage and to the clinical phenotype of the syndrome; it may also represent a pharmacological target to improve patient care and develop new cancer therapies.
| S-EPMC3090195 | biostudies-literature
Project description:In response to various environmental stresses, the stress-responsive MAPKs p38 and JNK are activated and phosphorylate ATF2 and c-Jun transcription factors, thereby affecting cell-fate decision. Targeted gene disruption studies have established that JNK-c-Jun signaling plays a vital role in stress-induced apoptosis. The oncogenic phosphatase Wip1 acts as an important regulator in DNA damage pathway by dephosphorylating a spectrum of proteins including p53, p38, Chk1, Chk2, and ATM. In this study we show that Wip1 negatively regulates the activation of MKK4-JNK-c-Jun signaling during stress-induced apoptosis. The loss of Wip1 function sensitizes mouse embryonic fibroblasts to stress-induced apoptosis via the activation of both p38-ATF2 and JNK-c-Jun signaling. Here we reveal that Wip1 has dual roles in alternatively regulating stress- and DNA damage-induced apoptosis through p38/JNK MAPKs and p38/p53-dependent pathways, respectively. Our results point to Wip1 as a general regulator of apoptosis, which further supports its role in tumorigenesis.
| S-EPMC2719383 | biostudies-literature
Project description:Fanconi anemia (FA) is a multigenic disease of bone marrow failure and cancer susceptibility stemming from a failure to remove DNA crosslinks and other chromosomal lesions. Within the FA DNA damage response pathway, DNA-dependent monoubiquitinaton of FANCD2 licenses downstream events, while timely FANCD2 deubiquitination serves to extinguish the response. Here, we show with reconstituted biochemical systems, which we developed, that efficient FANCD2 deubiquitination by the USP1-UAF1 complex is dependent on DNA and DNA binding by UAF1. Surprisingly, we find that the DNA binding activity of the UAF1-associated protein RAD51AP1 can substitute for that of UAF1 in FANCD2 deubiquitination in our biochemical system. We also reveal the importance of DNA binding by UAF1 and RAD51AP1 in FANCD2 deubiquitination in the cellular setting. Our results provide insights into a key step in the FA pathway and help define the multifaceted role of the USP1-UAF1-RAD51AP1 complex in DNA damage tolerance and genome repair.
| S-EPMC6599204 | biostudies-literature