Browsing by Subject "Ubiquitination"

Now showing 1 - 6 of 6
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    Akt Phosphorylates the Transcriptional Repressor Bmi1 to Block Its Effects on the Tumor-Suppressing Ink4a-Arf Locus
    (American Association for the Advancement of Science, 2012-10-23) Liu, Yan; Liu, Fan; Yu, Hao; Zhao, Xinyang; Sashida, Goro; Deblasio, Anthony; Harr, Michael; She, Qing-Bai; Chen, Zhenbang; Lin, Hui-Kuan; Di Giandomenico, Silvana; Elf, Shannon E.; Yang, Youyang; Miyata, Yasuhiko; Huang, Gang; Menendez, Silvia; Mellinghoff, Ingo K.; Rosen, Neal; Pandolfi, Pier Paolo; Hedvat, Cyrus V.; Nimer, Stephen D.; Pediatrics, School of Medicine
    The Polycomb group protein Bmi1 is a transcriptional silencer of the Ink4a-Arf locus, which encodes the cell cycle regulator p16(Ink4a) and the tumor suppressor p19(Arf). Bmi1 plays a key role in oncogenesis and stem cell self-renewal. We report that phosphorylation of human Bmi1 at Ser³¹⁶ by Akt impaired its function by triggering its dissociation from the Ink4a-Arf locus, which resulted in decreased ubiquitylation of histone H2A and the inability of Bmi1 to promote cellular proliferation and tumor growth. Moreover, Akt-mediated phosphorylation of Bmi1 also inhibited its ability to promote self-renewal of hematopoietic stem and progenitor cells. Our study provides a mechanism for the increased abundance of p16(Ink4a) and p19(Arf) seen in cancer cells with an activated phosphoinositide 3-kinase to Akt signaling pathway and identifies crosstalk between phosphorylation events and chromatin structure.
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    Depletion of PAK1 enhances Ubiquitin-mediated Survivin degradation in pancreatic β-cells
    (Taylor & Francis, 2013) Chen, Yi-Chun; Fueger, Patrick T.; Wang, Zhanxiang; Cellular and Integrative Physiology, School of Medicine
    Functional β-cell mass deficiency in diabetes results from imbalanced β-cell death and replication, and decreased PAK1 protein levels in human islets from donors with type 2 diabetes implicates a possible role for PAK1 in maintaining β-cell mass. Here, we aim to address the linkage between PAK1 and Survivin, a protein essential for β-cell replication. PAK1 knockout (KO) mouse islets exhibited decreased expression of Survivin protein. MIN6 β-cells with siRNA-mediated suppression of PAK1 also had decreased Survivin protein and exhibited an increased level of ubiquitinated-Survivin. However, no significant changes in Survivin mRNA were found in islets from PAK1 KO mice and PAK1-depleted MIN6 β-cells. The decreased Survivin level in MIN6 cells subjected to hyperglycemic stress was prevented by expression of exogenous PAK1. Moreover, overexpressing Survivin restored proliferation of β-cells that was impaired by the loss of PAK1. These data implicate a role for PAK1 in regulating Survivin protein stability in the β-cell and suggest PAK1 as a potential molecular target for the restoration of β-cell mass.
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    JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling
    (Wiley, 2020-07) Lü, Lanhai; Yakoumatos, Lan; Ren, Junling; Duan, Xiaoxian; Zhou, Huaxin; Gu, Zhen; Mohammed, Muddasir; Uriarte, Silvia M.; Liang, Shuang; Scott, David A.; Lamont, Richard J.; Wang, Huizhi; Medicine, School of Medicine
    Homeostasis between pro- and anti- inflammatory responses induced by bacteria is critical for the maintenance of health. In the oral cavity, proinflammatory mechanisms induced by pathogenic bacteria are well-established; however, the anti-inflammatory responses that act to restrain innate responses remain poorly characterized. Here, we demonstrate that infection with the periodontal pathogen P. gingivalis enhances the activity of JAK3 in innate immune cells, and subsequently phospho-inactivates Nedd4-2, a ubiquitin E3 ligase. In turn, Wnt3 ubiquitination is decreased, while total protein levels are enhanced, leading to a reduction in proinflammatory cytokine levels. In contrast, JAK3 inhibition or Wnt3a robustly enhances NF-κB activity and the production of proinflammatory cytokines in P. gingivalis-stimulated innate immune cells. Moreover, using gain- and loss-of-function approaches, we demonstrate that downstream molecules of Wnt3a signaling, including Dvl3 and β-catenin, are responsible for the negative regulatory role of Wnt3a. In addition, using an in vivo P. gingivalis-mediated periodontal disease model, we show that JAK3 inhibition enhances infiltration of inflammatory cells, reduces expression of Wnt3a and Dvl3 in P. gingivalis-infected gingival tissues, and increases disease severity. Together, our results reveal a new anti-inflammatory role for JAK3 in innate immune cells and show that the underlying signaling pathway involves Nedd4-2-mediated Wnt3a ubiquitination.
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    Proteomic Analysis of Nuclear Hepatitis B Virus Relaxed Circular DNA-Associated Proteins Identifies UV-Damaged DNA Binding Protein as a Host Factor Involved in Covalently Closed Circular DNA Formation
    (American Society for Microbiology, 2022) Marchetti, Alexander L.; Zhang, Hu; Kim, Elena S.; Yu, Xiaoyang; Jang, Sunbok; Wang, Mu; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the coprecipitated proteins by mass spectrometry. DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported short hairpin RNA (shRNA) screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex, and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of the DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Transcomplementation of wild-type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA binding, DDB1 binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE: Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified several host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNA interference (RNAi) screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay, we discovered a novel relationship between the UV-DDB complex and cccDNA formation, providing a proof of concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.
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    The DNA repair component Metnase regulates Chk1 stability
    (Springer Nature, 2014-07-09) Williamson, Elizabeth A.; Wu, Yuehan; Singh, Sudha; Byrne, Michael; Wray, Justin; Lee, Suk-Hee; Nickoloff, Jac A.; Hromas, Robert; Biochemistry and Molecular Biology, School of Medicine
    Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest.
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    Ubiquitination and degradation of SUMO1 by small-molecule degraders extends survival of mice with patient-derived tumors
    (AAAS, 2021) Bellail, Anita C.; Jin, Hong Ri; Lo, Ho-Yin; Jung, Sung Han; Hamdouchi, Chafiq; Kim, Daeho; Higgins, Ryan K.; Blanck, Maximilian; le Sage, Carlos; Cross, Benedict C. S.; Li, Jing; Mosley, Amber L.; Wijeratne, Aruna B.; Jiang, Wen; Ghosh, Manali; Zhao, Yin Quan; Hauck, Paula M.; Shekhar, Anantha; Hao, Chunhai; Pathology and Laboratory Medicine, School of Medicine
    Discovery of small-molecule degraders that activate ubiquitin ligase–mediated ubiquitination and degradation of targeted oncoproteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cancer cell–based drug screen of the NCI drug-like compounds library that enabled identification of small-molecule degraders of the small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies of analogs of the hit compound CPD1 led to identification of a lead compound HB007 with improved properties and anticancer potency in vitro and in vivo. A genome-scale CRISPR-Cas9 knockout screen identified the substrate receptor F-box protein 42 (FBXO42) of cullin 1 (CUL1) E3 ubiquitin ligase as required for HB007 activity. Using HB007 pull-down proteomics assays, we pinpointed HB007’s binding protein as the cytoplasmic activation/proliferation-associated protein 1 (CAPRIN1). Biolayer interferometry and compound competitive immunoblot assays confirmed the selectivity of HB007’s binding to CAPRIN1. When bound to CAPRIN1, HB007 induced the interaction of CAPRIN1 with FBXO42. FBXO42 then recruited SUMO1 to the CAPRIN1-CUL1-FBXO42 ubiquitin ligase complex, where SUMO1 was ubiquitinated in several of human cancer cells. HB007 selectively degraded SUMO1 in patient tumor–derived xenografts implanted into mice. Systemic administration of HB007 inhibited the progression of patient-derived brain, breast, colon, and lung cancers in mice and increased survival of the animals. This cancer cell–based screening approach enabled discovery of a small-molecule degrader of SUMO1 and may be useful for identifying other small-molecule degraders of oncoproteins.