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Met原癌基因在肝脏中的一个主要功能是抑制肝细胞的凋亡。我们认为Met引发抗凋亡作用的一种机制是通过Met与促死亡细胞表面受体Fas之间的相互作用，其最终结果是Met隔离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直接抑制凋亡通路的刽子手，即效应caspase (casepase- 3和caspase-7)，通过底物自杀机制利用Met蛋白的细胞质c端尾部。这些相互作用抑制凋亡指令，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相互作用所需的结构域。我们将利用转基因和基因敲除小鼠模型探讨Met对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Aim 2中，我们将验证Met是效应caspase（如caspase-3）在细胞凋亡过程中破坏性作用的关键靶点这一假设。这一假设是基于我们的发现，即Met对细胞存活至关重要，因为在Met的酪氨酸激酶激活区域存在一个完美的半胱天冬酶切割位点。重要的是，这个酪氨酸激酶激活区域（在我们已经确定的假定的半胱天冬酶切割位点）的种系和散发性突变已经被报道。我们将研究这些突变使Met对半胱天冬酶的切割难以耐受，从而导致细胞抵抗凋亡并导致恶性转化的假设。在Aim 3中，我们提出验证Met蛋白已经进化出一种新的机制来逃避/抑制凋亡途径的刽子手，即效应半胱天冬酶的假设。我们认为Met对caspase的抑制是通过一种底物自杀机制实现的，该机制涉及Met分子的胞内细胞质端。在这个区域，我们发现了八肽序列DNADDEVD。由于该肽序列包含串联的完美效应caspase切割位点（即caspase-3），我们提出在细胞凋亡过程中，该肽序列被这些caspase切割，从而形成四肽DEVD，这是一种众所周知的caspase-3和caspase-7的有效抑制剂。在Aim 4中，我们将验证met基因在细胞应激反应中被诱导以保护细胞免于凋亡的假设，并且是NF-kappaB上调的靶标，NF-kappaB是一种已知对胚胎发育期间肝细胞存活至关重要的转录因子。