Mechanisms of met-Induced Hepatocytes Survival

met 诱导肝细胞存活的机制

• 批准号: 6472032
• 负责人: Reza Zarnegar
• 金额: $ 28.54万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6472032
• 关键词: apoptosis; biological signal transduction; cysteine endopeptidases; cytokine receptors; cytoprotection; enzyme inhibitors; fluorescence resonance energy transfer; gene induction /repression; gene interaction; gene targeting; genetically modified animals; hepatocellular carcinoma; homeostasis; immunoprecipitation; laboratory mouse; liver cells; liver failure; nuclear factor kappa beta; protein degradation; protein structure function; protein tyrosine kinase; protooncogene; renal cell carcinoma; tissue /cell culture; yeast two hybrid system

Based on the results of our recent experiments, we hypothesize that one major function of Met protooncogene in the liver is to inhibit apoptosis in hepatocytes. We believe that one mechanism by which Met elicits an anti-apoptotic effect is via an interaction between Met and the death promoting cell surface receptor Fas, the net result of which is sequestration of Fas by Met and inhibition of the initiation of the Fas-mediated apoptotic pathway. Another novel mechanism by which Met may modulate apoptosis involves direct inhibition of the executioners of the apoptotic pathway, namely the effector caspases (casepase- 3 and caspase-7), by a substrate suicide mechanism employing the cytoplasmic c-terminal tail of the Met protein. These interactions squelch the apoptotic command and thus promote cell survival. In Aim 1, we will determine the nature of the Met-Fas interaction and will map the structural domains in Met and Fas that are required for their association. We will address the biological relevance of Fas sequestration by Met and its contribution to hepatocyte survival, liver development, hepatic homeostasis and transformation using transgenic and knock out mouse models. In Aim 2, we will test the hypothesis that Met is a key target of the destructive action of the effector caspases (such as caspase-3) during apoptosis. This hypothesis is based on our findings that Met is essential for cell survival and because a perfect caspase cleavage site is present in Met's tyrosine kinase activation domain. Importantly, germline and sporadic mutations in this tyrosine kinase activation domain (in the putative caspase cleavage site that we have identified) have been reported. We will investigate the hypothesis that these mutations make Met refractory to caspase cleavage thus causing cells to be resistant to apoptosis and contributing to malignant transformation. In Aim 3, we propose to test the hypothesis that Met protein has evolved a novel mechanism to elude/inhibit the executioners of the apoptotic pathway, namely the effector caspases. We propose that caspase inhibition by Met is achieved through a substrate suicide mechanism involving the intracellular cytoplasmic end of the Met molecule. In this region, we have discovered the octapeptide sequence DNADDEVD. Since this peptide sequence harbors tandem perfect effector caspase cleavage sites (i.e. for caspase-3), we propose that during apoptosis this peptide sequence is cleaved by these caspases resulting in the formation of the tetrapeptide DEVD, a well-known potent inhibitor of caspase-3 and caspase-7. In Aim 4, we will test the hypothesis that the met gene is induced in response to cellular stress to protect cells from apoptosis and is a target of upregulation of NF-kappaB, a transcription factor known to be essential for hepatocyte survival during embryonic development.

根据我们最近的实验结果，我们假设肝脏中 Met 原癌基因的一项主要功能是抑制肝细胞凋亡。 我们认为，Met 引发抗凋亡作用的一种机制是通过 Met 与促进死亡的细胞表面受体 Fas 之间的相互作用，其最终结果是 Met 螯合 Fas 并抑制 Fas 介导的凋亡途径的启动。 Met 调节细胞凋亡的另一种新机制涉及通过利用 Met 蛋白的细胞质 C 末端尾部的底物自杀机制，直接抑制细胞凋亡途径的执行者，即效应器 caspase（caspase-3 和 caspase-7）。 这些相互作用抑制了细胞凋亡命令，从而促进细胞存活。 在目标 1 中，我们将确定 Met-Fas 相互作用的性质，并绘制 Met 和 Fas 关联所需的结构域。 我们将使用转基因和敲除小鼠模型来探讨 Met 封存 Fas 的生物学相关性及其对肝细胞存活、肝脏发育、肝脏稳态和转化的贡献。 在目标 2 中，我们将检验这样的假设：Met 是细胞凋亡过程中效应 caspase（例如 caspase-3）破坏作用的关键靶标。 这一假设基于我们的发现，即 Met 对于细胞生存至关重要，并且因为 Met 的酪氨酸激酶激活域中存在完美的 caspase 裂解位点。 重要的是，已经报道了该酪氨酸激酶激活域（在我们已经确定的假定的 caspase 裂解位点）中的种系和散发突变。 我们将研究以下假设：这些突变使 Met 对 caspase 切割产生抵抗，从而导致细胞抵抗细胞凋亡并导致恶性转化。 在目标 3 中，我们建议检验这样的假设：Met 蛋白已经进化出一种新的机制来逃避/抑制细胞凋亡途径的执行者，即效应器半胱天冬酶。 我们提出 Met 抑制 caspase 是通过涉及 Met 分子的细胞内胞质末端的底物自杀机制来实现的。 在这个区域，我们发现了八肽序列DNADDEVD。 由于该肽序列具有串联完美效应器 caspase 裂解位点（即 caspase-3），因此我们提出在细胞凋亡过程中，该肽序列被这些 caspase 裂解，导致形成四肽 DEVD，这是一种众所周知的 caspase-3 和 caspase-7 的有效抑制剂。 在目标 4 中，我们将检验这样的假设：met 基因是响应细胞应激而诱导的，以保护细胞免于凋亡，并且是 NF-kappaB 上调的目标，NF-kappaB 是一种已知对胚胎发育过程中肝细胞存活至关重要的转录因子。