The Experts below are selected from a list of 2124 Experts worldwide ranked by ideXlab platform
Jiayu Liao - One of the best experts on this subject based on the ideXlab platform.
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a novel robust quantitative forster resonance energy transfer assay for protease SENP2 kinetics determination against its all natural substrates
Molecular BioSystems, 2015Co-Authors: Yan Liu, Yali Shen, Shasha Zheng, Jiayu LiaoAbstract:SUMOylation (the process of adding the SUMO [small ubiquitin-like modifier] to substrates) is an important post-translational modification of critical proteins in multiple processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate the SUMO from its substrate. Determining the kinetics of SENPs is important for understanding their activities. Forster resonance energy transfer (FRET) technology has been widely used in biomedical research and is a powerful tool for elucidating protein interactions. In this paper we report a novel quantitative FRET-based protease assay for SENP2 endopeptidase activity that accounts for the self-fluorescent emissions of the donor (CyPet) and the acceptor (YPet). The kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP2 toward pre-SUMO1/2/3, were obtained by this novel design. Although we use SENP2 to demonstrate our method, the general principles of this quantitative FRET-based protease kinetic determination can be readily applied to other proteases.
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engineering pre sumo4 as efficient substrate of SENP2
Protein Engineering Design & Selection, 2014Co-Authors: Yan Liu, Chris A Kieslich, Dimitrios Morikis, Jiayu LiaoAbstract:SUMOylation, one of the most important protein post-translational modifications, plays critical roles in a variety of physiological and pathological processes. SENP (Sentrin/SUMO-specific protease), a family of SUMO-specific proteases, is responsible for the processing of pre-SUMO and removal of SUMO from conjugated substrates. SUMO4, the latest discovered member in the SUMO family, has been found as a type 1 diabetes susceptibility gene and its maturation is not understood so far. Despite the 14 amino acid differences between pre-SUMO4 and SUMO2, pre-SUMO4 is not processed by SENP2 but pre-SUMO2 does. A novel interdisciplinary approach involving computational modeling and a FRET-based protease assay was taken to engineer pre-SUMO4 as a substrate of SENP2. Given the difference in net charge between pre-SUMO4 and pre-SUMO2, the computational framework analysis of electrostatic similarities of proteins was applied to determine the contribution of each ionizable amino acid in a model of SENP2-(pre-SUMO4) binding, and to propose pre-SUMO4 mutations. The specificities of the SENP2 toward different pre-SUMO4 mutants were determined using a quantitative FRET assay by characterizing the catalytic efficiencies (kcat/KM). A single amino acid mutation made pre-SUMO4 amenable to SENP2 processing and a combination of two amino acid mutations made it highly accessible as SENP2 substrate. The combination of the two approaches provides a powerful protein engineering tool for future SUMOylation studies.
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quantitative fret forster resonance energy transfer analysis for senp1 protease kinetics determination
Journal of Visualized Experiments, 2013Co-Authors: Yan Liu, Jiayu LiaoAbstract:Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle (1,3,6,7). The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate (8-13). However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs. Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (9,10,14,15). The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate. We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate (16). We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 ± 0.55) x10(7) M(-1)s(-1) of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.
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quantitative forster resonance energy transfer analysis for kinetic determinations of sumo specific protease
Analytical Biochemistry, 2012Co-Authors: Yang Song, Vipul Madahar, Jiayu LiaoAbstract:Abstract Forster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet–SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.
Jinke Cheng - One of the best experts on this subject based on the ideXlab platform.
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SENP2 plcβ4 signaling regulates neurogenesis through the maintenance of calcium homeostasis
Cell Death & Differentiation, 2021Co-Authors: Xu Chen, Jinke Cheng, Xinyi Yang, Yuhong Zhang, Yuanyuan Qin, Zhengcao Xing, Yajie Shen, Edward T H YehAbstract:Neurogenesis plays a critical role in brain physiology and behavioral performance, and defective neurogenesis leads to neurological and psychiatric disorders. Here, we show that PLCβ4 expression is markedly reduced in SENP2-deficient cells and mice, resulting in decreased IP3 formation and altered intracellular calcium homeostasis. PLCβ4 stability is regulated by the SUMO-dependent ubiquitin-mediated proteolytic pathway, which is catalyzed by PIAS2α and RNF4. SUMOylated PLCβ4 is transported to the nucleus through Nup205- and RanBP2-dependent pathways and regulates nuclear signaling. Furthermore, dysregulated calcium homeostasis induced defects in neurogenesis and neuronal viability in SENP2-deficient mice. Finally, SENP2 and PLCβ4 are stimulated by starvation and oxidative stress, which maintain calcium homeostasis regulated neurogenesis. Our findings provide mechanistic insight into the critical roles of SENP2 in the regulation of PLCβ4 SUMOylation, and the involvement of SENP2-PLCβ4 axis in calcium homeostasis regulated neurogenesis under stress.
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SENP2 Suppresses Necdin Expression to Promote Brown Adipocyte Differentiation
Cell reports, 2019Co-Authors: Qiuli Liang, Yalan Chen, Quan Zheng, Yong Zuo, Jinke ChengAbstract:Summary Brown adipose tissue (BAT) is a thermogenic organ that maintains body temperature and energy homeostasis. Transcriptional regulation plays an important role in the program of brown adipogenesis. However, it remains unclear how the transcriptional events are controlled in this program. In this study, we analyze an SENP2 BAT conditional knockout mouse model and find that SENP2-mediated de-SUMOylation is essential for BAT development. SENP2 catalyzes de-SUMOylation of cAMP response element-binding protein (CREB) to suppress Necdin expression, which induces brown adipocyte differentiation and brown adipogenesis. Mechanistically, we find that SUMOylation enhances CREB interaction with serine/threonine protein phosphatase 2A (PP2A) to de-phosphorylate CREB, which activates Necdin transcription. SENP2 deficiency enhances the expression of Necdin to inhibit brown adipocyte differentiation. Therefore, we reveal a crucial role of SENP2-mediated de-SUMOylation of CREB in suppression of Necdin expression during brown adipose development and brown adipogenesis.
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SENP2 regulates adipose lipid storage by de sumoylation of setdb1
Journal of Molecular Cell Biology, 2018Co-Authors: Quan Zheng, Xiuzhi Wang, Jiqiu Wang, Xian Huang, Tianshi Wang, Yiping Wang, Yalan Chen, Jinke ChengAbstract:: One major function of adipocytes is to store excess energy in the form of triglycerides. Insufficient adipose lipid storage is associated with many pathological conditions including hyperlipidemia, insulin resistance, and type 2 diabetes. In this study, we observed the overexpression of SUMO-specific protease 2 (SENP2) in adipose tissues during obesity. Adipocyte SENP2 deficiency resulted in less adipose lipid storage accompanied by an ectopic fat accumulation and insulin resistance under high-fat diet feeding. We further found that SET domain bifurcated 1 (Setdb1) was a SUMOylated protein and that SUMOylation promoted Setdb1 occupancy on the promoter locus of Pparg and Cebpa genes to suppress their expressions by H3K9me3. SENP2 could suppress Setdb1 function by de-SUMOylation. In adipocyte SENP2-deficiency mice, accumulation of the SUMOylated Setdb1 suppressed the expression of Pparg and Cebpa genes as well as lipid metabolism-related target genes, which would decrease the ability of lipid storage in adipocytes. These results revealed the crucial role of SENP2-Setdb1 axis in controlling adipose lipid storage.
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mitotic phosphorylation of senp3 regulates desumoylation of chromosome associated proteins and chromosome stability
Cancer Research, 2018Co-Authors: Bo Wei, Qiuju Fan, Chao Huang, Bin Liu, Yang Wang, Nansong Xia, Guoqiang Chen, Jinke ChengAbstract:Progression of mitotic cell cycle and chromosome condensation and segregation are controlled by posttranslational protein modifications such as phosphorylation and SUMOylation. However, how SUMO isopeptidases (SENP) regulate cell mitotic procession is largely unknown. Here, we demonstrate that precise phosphorylation of SENP3 during mitosis suppresses SENP3 deSUMOylation activity towards chromosome-associated proteins, including topoisomerase IIα (TopoIIα). Cyclin B-dependent kinases 1 and protein phosphatase 1α were identified as the kinase and phosphatase in control of mitotic SENP3 phosphorylation, respectively. SENP3 phosphorylation decreased its interaction with TopoIIα, resulting in reduced SENP3 deSUMOylation activity on TopoIIα. Furthermore, we observed mitotic arrest, increased chromosome instability, and promotion of tumorigenesis in cells expressing a nonphosphorylatable SENP3 mutant. These data show that SENP3 phosphorylation plays a crucial role in regulating the SUMOylation of chromosome-associated proteins and chromosome stability in mitosis.Significance: Phosphorylation of SENP3 regulates SUMOylation of chromosome-associated proteins to maintain genomic stability during mitosis. Cancer Res; 78(9); 2171-8. ©2018 AACR.
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senp1 promotes proliferation of clear cell renal cell carcinoma through activation of glycolysis
Oncotarget, 2016Co-Authors: Baijun Dong, Xunlei Kang, Wei Xue, Yujing Gao, Hongchang Gao, Jin Zhang, Hua Guo, Mingjian J You, Jinke ChengAbstract:// Baijun Dong 1, * , Yujing Gao 2, * , Xunlei Kang 3, * , Hongchang Gao 4, * , Jin Zhang 1 , Hua Guo 5, 7 , Mingjian J You 5, 6 , Wei Xue 1 , Jinke Cheng 3 , Yiran Huang 1 1 Department of Urology, School of Medicine, Renji Hospital, Shanghai Jiao Tong University, Shanghai, China 2 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Department of Biochemistry and Molecular Biology, Ningxia Medical University, Yinchuan, Ningxia, China 3 Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China 4 School of Pharmacy, Wenzhou Medical College, Wenzhou, China 5 Department of Hematopathology, University of Texas MD Anderson Cancer Center, Houston, USA 6 The Graduate School of Biomedical Science, University of Texas MD Anderson Cancer Center, Houston, USA 7 Current address: North Shore LIJ Health System, New York, USA * These authors contributed equally to this work Correspondence to: Yiran Huang, email: huangyiran@renji.com Jinke Cheng, email: jkcheng@shsmu.edu.cn Keywords: SENP1, glycolysis, HIF-1α, clear cell renal cell carcinoma Received: May 29, 2016 Accepted: October 03, 2016 Published: October 12, 2016 ABSTRACT Metabolic shift toward aerobic glycolysis is a fundamental element contributing to the development and progression of clear cell renal cell carcinoma (ccRCC). We and others previously observed enhanced glycolysis and diminished tricarboxylic acid (TCA) cycle activity in ccRCC tissue. Here, by integrated gene expression and metabolomic analyses of 36 matched pairs of tumor and adjacent normal tissues, we showed that expression of Sentrin/SUMO-specific protease 1 (SENP1) is positively associated with glycolysis levels in ccRCC. Moreover, SENP1 knockdown in RCC4/VHL cells downregulated expression of key glycolytic enzymes under normoxic and hypoxic conditions and inhibited cell proliferation under hypoxic conditions, possibly due to ineffective deSUMOylation and stablization of Hif-1α related to the SENP-1 deficiency. Finally, SENP1 expression correlated positively with tumor pathological grade and was an indicator of poor overall survival and advanced tumor progression in ccRCC. Altered VHL gene function is found in 60–90% ccRCC cases of ccRCC, but therapies targeting VHL-related signaling pathways have been ineffective, spurring exploration of alternative pathological signaling events. Our results provide a possible mechanistic explanation for the role of SENP1 in the initiation and development of ccRCC with normal VHL activity, and identifies SENP1 as a potential treatment target for the disease.
Yan Liu - One of the best experts on this subject based on the ideXlab platform.
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a novel robust quantitative forster resonance energy transfer assay for protease SENP2 kinetics determination against its all natural substrates
Molecular BioSystems, 2015Co-Authors: Yan Liu, Yali Shen, Shasha Zheng, Jiayu LiaoAbstract:SUMOylation (the process of adding the SUMO [small ubiquitin-like modifier] to substrates) is an important post-translational modification of critical proteins in multiple processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate the SUMO from its substrate. Determining the kinetics of SENPs is important for understanding their activities. Forster resonance energy transfer (FRET) technology has been widely used in biomedical research and is a powerful tool for elucidating protein interactions. In this paper we report a novel quantitative FRET-based protease assay for SENP2 endopeptidase activity that accounts for the self-fluorescent emissions of the donor (CyPet) and the acceptor (YPet). The kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP2 toward pre-SUMO1/2/3, were obtained by this novel design. Although we use SENP2 to demonstrate our method, the general principles of this quantitative FRET-based protease kinetic determination can be readily applied to other proteases.
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engineering pre sumo4 as efficient substrate of SENP2
Protein Engineering Design & Selection, 2014Co-Authors: Yan Liu, Chris A Kieslich, Dimitrios Morikis, Jiayu LiaoAbstract:SUMOylation, one of the most important protein post-translational modifications, plays critical roles in a variety of physiological and pathological processes. SENP (Sentrin/SUMO-specific protease), a family of SUMO-specific proteases, is responsible for the processing of pre-SUMO and removal of SUMO from conjugated substrates. SUMO4, the latest discovered member in the SUMO family, has been found as a type 1 diabetes susceptibility gene and its maturation is not understood so far. Despite the 14 amino acid differences between pre-SUMO4 and SUMO2, pre-SUMO4 is not processed by SENP2 but pre-SUMO2 does. A novel interdisciplinary approach involving computational modeling and a FRET-based protease assay was taken to engineer pre-SUMO4 as a substrate of SENP2. Given the difference in net charge between pre-SUMO4 and pre-SUMO2, the computational framework analysis of electrostatic similarities of proteins was applied to determine the contribution of each ionizable amino acid in a model of SENP2-(pre-SUMO4) binding, and to propose pre-SUMO4 mutations. The specificities of the SENP2 toward different pre-SUMO4 mutants were determined using a quantitative FRET assay by characterizing the catalytic efficiencies (kcat/KM). A single amino acid mutation made pre-SUMO4 amenable to SENP2 processing and a combination of two amino acid mutations made it highly accessible as SENP2 substrate. The combination of the two approaches provides a powerful protein engineering tool for future SUMOylation studies.
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quantitative fret forster resonance energy transfer analysis for senp1 protease kinetics determination
Journal of Visualized Experiments, 2013Co-Authors: Yan Liu, Jiayu LiaoAbstract:Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle (1,3,6,7). The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate (8-13). However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs. Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (9,10,14,15). The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate. We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate (16). We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 ± 0.55) x10(7) M(-1)s(-1) of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.
Marxa L Figueiredo - One of the best experts on this subject based on the ideXlab platform.
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abstract lb 074 examining senp localization in hek 293 cells using mg 132 and cycloheximide
Cancer Research, 2018Co-Authors: Michael Robinson, Charles Samuel Umbaugh, Marxa L FigueiredoAbstract:Post translational modifications are changes to proteins that occur during or after synthesis by the ribosome. Often these modifications are necessary to form mature proteins. SUMOylation ( S mall U biquitin-like M odifier, SUMO) is a type of post translational modification that has a connection to a protein, Laminin Receptor, that contributes to the aggressiveness and proliferation of cancer. SUMO proteases, called SENPs, remove SUMO groups from SUMOylated proteins. Because Laminin Receptor may be a SUMOylated protein and is in the nucleus, we wanted to first explore how SENP-2 and SENP-3 localize under various cellular conditions. To understand the behavior of SENPs under cellular stress, we transfected human embryonic kidney cells, called HEK-293 cells, with SENP-2 or SENP-3 tagged to the fluorescent protein EGFP. We treated the cells with MG-132, a proteasome inhibitor, or with cycloheximide, a protein synthesis inhibitor. We used live imaging to qualitatively understand how SENP-2 and SENP-3 localize in the cell in normal conditions and under cell stress. Then, we looked for differences in cytosolic and nuclear partitioning of SENP-2 and SENP-3 by western blot. The exposure of SENPs to the two inhibitors helped us to understand the behavior of the SUMO proteases between the nucleus and cytoplasm. Citation Format: Michael Robinson, Charles S. Umbaugh, Marxa L. Figueiredo. Examining SENP Localization In HEK-293 Cells Using MG-132 And Cycloheximide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-074.
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abstract lb 074 examining senp localization in hek 293 cells using mg 132 and cycloheximide
Cancer Research, 2018Co-Authors: Michael Robinson, Charles Samuel Umbaugh, Marxa L FigueiredoAbstract:Post translational modifications are changes to proteins that occur during or after synthesis by the ribosome. Often these modifications are necessary to form mature proteins. SUMOylation ( S mall U biquitin-like M odifier, SUMO) is a type of post translational modification that has a connection to a protein, Laminin Receptor, that contributes to the aggressiveness and proliferation of cancer. SUMO proteases, called SENPs, remove SUMO groups from SUMOylated proteins. Because Laminin Receptor may be a SUMOylated protein and is in the nucleus, we wanted to first explore how SENP-2 and SENP-3 localize under various cellular conditions. To understand the behavior of SENPs under cellular stress, we transfected human embryonic kidney cells, called HEK-293 cells, with SENP-2 or SENP-3 tagged to the fluorescent protein EGFP. We treated the cells with MG-132, a proteasome inhibitor, or with cycloheximide, a protein synthesis inhibitor. We used live imaging to qualitatively understand how SENP-2 and SENP-3 localize in the cell in normal conditions and under cell stress. Then, we looked for differences in cytosolic and nuclear partitioning of SENP-2 and SENP-3 by western blot. The exposure of SENPs to the two inhibitors helped us to understand the behavior of the SUMO proteases between the nucleus and cytoplasm. Citation Format: Michael Robinson, Charles S. Umbaugh, Marxa L. Figueiredo. Examining SENP Localization In HEK-293 Cells Using MG-132 And Cycloheximide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-074.
Edward T H Yeh - One of the best experts on this subject based on the ideXlab platform.
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SENP2 plcβ4 signaling regulates neurogenesis through the maintenance of calcium homeostasis
Cell Death & Differentiation, 2021Co-Authors: Xu Chen, Jinke Cheng, Xinyi Yang, Yuhong Zhang, Yuanyuan Qin, Zhengcao Xing, Yajie Shen, Edward T H YehAbstract:Neurogenesis plays a critical role in brain physiology and behavioral performance, and defective neurogenesis leads to neurological and psychiatric disorders. Here, we show that PLCβ4 expression is markedly reduced in SENP2-deficient cells and mice, resulting in decreased IP3 formation and altered intracellular calcium homeostasis. PLCβ4 stability is regulated by the SUMO-dependent ubiquitin-mediated proteolytic pathway, which is catalyzed by PIAS2α and RNF4. SUMOylated PLCβ4 is transported to the nucleus through Nup205- and RanBP2-dependent pathways and regulates nuclear signaling. Furthermore, dysregulated calcium homeostasis induced defects in neurogenesis and neuronal viability in SENP2-deficient mice. Finally, SENP2 and PLCβ4 are stimulated by starvation and oxidative stress, which maintain calcium homeostasis regulated neurogenesis. Our findings provide mechanistic insight into the critical roles of SENP2 in the regulation of PLCβ4 SUMOylation, and the involvement of SENP2-PLCβ4 axis in calcium homeostasis regulated neurogenesis under stress.
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disturbed flow activated p90rsk kinase accelerates atherosclerosis by inhibiting SENP2 function
Journal of Clinical Investigation, 2015Co-Authors: Kyungsun Heo, Edward T H Yeh, Hannah J Cushman, Carolyn J Giancursio, Eugene Chang, Chang Hoon Woo, Mark A Sullivan, Jack Taunton, Keigi FujiwaraAbstract:Disturbed blood flow (d-flow) causes endothelial cell (EC) dysfunction, leading to atherosclerotic plaque formation. We have previously shown that d-flow increases SUMOylation of p53 and ERK5 through downregulation of sentrin/SUMO-specific protease 2 (SENP2) function; however, it is not known how SENP2 itself is regulated by d-flow. Here, we determined that d-flow activated the serine/threonine kinase p90RSK, which subsequently phosphorylated threonine 368 (T368) of SENP2. T368 phosphorylation promoted nuclear export of SENP2, leading to downregulation of eNOS expression and upregulation of proinflammatory adhesion molecule expression and apoptosis. In an LDLR-deficient murine model of atherosclerosis, EC-specific overexpression of p90RSK increased EC dysfunction and lipid accumulation in the aorta compared with control animals; however, these pathologic changes were not observed in atherosclerotic mice overexpressing dominant negative p90RSK (DN-p90RSK). Moreover, depletion of SENP2 in these mice abolished the protective effect of DN-p90RSK overexpression. We propose that p90RSK-mediated SENP2-T368 phosphorylation is a master switch in d-flow–induced signaling, leading to EC dysfunction and atherosclerosis.
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nf κb induction of the sumo protease SENP2 a negative feedback loop to attenuate cell survival response to genotoxic stress
Molecular Cell, 2011Co-Authors: Moon Hee Lee, Edward T H Yeh, Angela M Mabb, Grace Gill, Shigeki MiyamotoAbstract:Activation of NF-κB, pivotal for immunity and oncogenesis, is tightly controlled by multiple feedback mechanisms. In response to DNA damage, SUMOylation of NEMO (NF-κB essential modulator) is critical for NF-κB activation; however, the SUMO proteases and feedback mechanisms involved remain unknown. Here we show that among the six known Sentrin/SUMO-specific proteases (SENPs), only SENP2 can efficiently associate with NEMO, deSUMOylate NEMO, and inhibit NF-κB activation induced by DNA damage. We further show that NF-κB induces SENP2 (and SENP1) transcription selectively in response to genotoxic stimuli, which involves ataxia telangiectasia mutated (ATM)-dependent histone methylation of SENP2 promoter κB regions and NF-κB recruitment. SENP2 null cells display biphasic NEMO SUMOylation and activation of IKK and NF-κB, and higher resistance to DNA damage-induced cell death. Our study establishes a self-attenuating feedback mechanism selective to DNA damage-induced signaling to limit NF-κB-dependent cell survival responses.
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sumo specific protease 2 is essential for suppression of polycomb group protein mediated gene silencing during embryonic development
Molecular Cell, 2010Co-Authors: Xunlei Kang, Jinke Cheng, Yong Zuo, Qi Wang, Yanqiong Zou, Robert J Schwartz, Edward T H YehAbstract:SUMO-specific protease 2 (SENP2) has a broad de-SUMOylation activity in vitro. However, the biological function of SENP2 is largely unknown. Here, we show that deletion of SENP2 gene in mouse causes defects in the embryonic heart and reduces the expression of Gata4 and Gata6, which are essential for cardiac development. SENP2 regulates transcription of Gata4 and Gata6 mainly through alteration of occupancy of Pc2/CBX4, a polycomb repressive complex 1 (PRC1) subunit, on its promoters. We demonstrate that Pc2/CBX4 is a target of SENP2 in vivo and that SUMOylation is essential for Pc2/CBX4-mediated PRC1 recruitment to methylated histone 3 at K27 (H3K27me3). In SENP2 null embryos, SUMOylated Pc2/CBX4 accumulates and Pc2/CBX4 occupancy on the promoters of PcG target genes is markedly increased, leading to repression of Gata4 and Gata6 transcription. Our results reveal a critical role for de-SUMOylation in the regulation of PcG target gene expression.
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senp3 is responsible for hif 1 transactivation under mild oxidative stress via p300 de sumoylation
The EMBO Journal, 2009Co-Authors: Chao Huang, Edward T H Yeh, Xuxu Sun, Yan Han, Yumei Wang, Shan Yan, Yuying Chen, Hui Cang, Guiying Shi, Jinke ChengAbstract:The physiological function of Sentrin/SUMO-specific proteases (SENPs) remains largely unexplored, and little is known about the regulation of SENPs themselves. Here, we show that a modest increase of reactive oxygen species (ROS) regulates SENP3 stability and localization. We found that SENP3 is continuously degraded through the ubiquitin-proteasome pathway under basal condition and that ROS inhibit this degradation. Furthermore, ROS causes SENP3 to redistribute from the nucleoli to the nucleoplasm, allowing it to regulate nuclear events. The stabilization and redistribution of SENP3 correlate with an increase in the transcriptional activity of the hypoxia-inducing factor-1 (HIF-1) under mild oxidative stress. ROS-enhanced HIF-1 transactivation is blocked by SENP3 knockdown. The de-SUMOylating activity of SENP3 is required for ROS-induced increase of HIF-1 transactivation, but the true substrate of SENP3 is the co-activator of HIF-1α, p300, rather than HIF-1α itself. Removing SUMO2/3 from p300 enhances its binding to HIF-1α. In vivo nude mouse xenografts overexpressing SENP3 are more angiogenic. Taken together, our results identify SENP3 as a redox sensor that regulates HIF-1 transcriptional activity under oxidative stress through the de-SUMOylation of p300.
