秦正红：DRAM1 regulates autophagy flux and Bid-mediated cell death via lysosomes
秦正红，博士，教授，神经药理专业博士生导师。1994年在美国宾州医学院研究生院获博士学位，先后在美国国家卫生研究院(NIH)及麻省总医院和哈佛大学医学院从事研究工作。2003年从哈佛大学引进，现为苏州大学医学部基础医学与生物科学学院科研中心实验室主任，中国药理学会生化药理学专业委员会委员，中国药理学会神经药理学专业委员会委员，美国神经科学学会会员。<Br><Br>Damage-regulated autophagy modulator1 (DRAM1), a novel TP53 target gene, is an evolutionarily conserved lysosomal protein and plays an essential role in TP53-dependent autophagy activation and apoptosis (Crighton et al, 2006). However, the mechanisms By which DRAM1 promotes autophagy and apoptosis are not clear. 3-Nitropropionic acid (3-NP) is an inhiBitor of mitochondrial respiratory complex II. Intrastriatal administration of 3-NP produces neuropathology resemBle to Huntington disease. 3-NP-induced neuronal death was involved in autophagy and apoptosis. In vitro studies with 3-NP in TP53 wt and null cells, 3-NP or CCCP increased the protein levels of DRAM1 in a TP53-dependent or independent manner. DRAM1 induction contriButed to 3-NP-induced autophagy activation. Knock-down of DRAM1 with siRNA inhiBited the activity of V-ATPase, acidification of lysosomes and activation of lysosomal proteases. Knock-down of DRAM1 reduced the clearance of autophagososmes. <Br> <Br> 3-NP also induced a transcription independent upregulation of BAX protein levels. Knock-down of DRAM1 suppressed the increase in BAX levels. Co-immunoprecipitation and pull-down studies revealed an interaction of DRAM1 and BAX protein. StaBly expression of exogenous DRAM1 increased the half-life of BAX. Upregulation of DRAM1 recruited BAX to lysosomes and induced cathepsin B-dependent cleavage of Bid and cytochrome c release. Knockdown of DRAM1, BAX or inhiBition of lysosomal enzymes reduced 3-NP-induced cytochrome c release and cell death. <Br> <Br> These data suggest that DRAM1 plays important roles in regulating autophagy flux and apoptosis. DRAM1 promotes autophagy flux through a mechanism involves activation of V-ATPase and enhances the acidification of lysosomes. DRAM1 promotes apoptosis via a mechanism involving recruitment of BAX to lysosomes to trigger cathepsin B-mediated Bid cleavage.
Generating B-lymphoBlastoid cell lines using Epstein Barr virus transformation.
Generating immortalized B-lymphoBlastoid cell lines via Epstein Barr virus transformation using the B95-8 EBV-infected and producing marmoset cell line.
从外周血中提取DraBek基因组DNA
DraBek genomic DNA extractions from peripheral Blood. Employing a novel DNA extraction procedure adapted from DraBek [Biomed. Papers 146(2), 37–39 (2002)] and Nasiri [Journal of Clinical LaBoratory Analysis 19:229–232 (2005)]for human peripheral Blood samples.
建立EB病毒转化B淋巴母细胞系
Generating B-lymphoBlastoid cell lines using Epstein Barr virus transformation. Generating immortalized B-lymphoBlastoid cell lines via Epstein Barr virus transformation using the B95-8 EBV-infected and producing marmoset cell line.
Western Blot Using The invitrogen NuPAGE Novex Bis-Tris MiniGel System(AuBin Penna.Ph.D)
Western Blot Using The invitrogen NuPAGE Novex Bis-Tris MiniGel System(AuBin Penna.Ph.D)
ImmunoBlot Analysis Sean Gallagher(UVP,LLC)and DeB Chakravart(Proteomic Center)
ImmunoBlot Analysis Sean Gallagher(UVP,LLC)and DeB Chakravart(Proteomic Center)
