Mechanisms of met-Induced Hepatocytes Survival

基于我们最近的实验结果，我们推测Met原癌基因在肝脏中的一个主要功能是抑制肝细胞凋亡。 我们认为，Met发挥抗凋亡作用的一种机制是通过Met与促死细胞表面受体Fas之间的相互作用，其净结果是Met螯合Fas并抑制Fas介导的凋亡途径的启动。 Met可调节细胞凋亡的另一种新机制涉及通过采用Met蛋白质的细胞质C-末端尾的底物自杀机制直接抑制细胞凋亡途径的执行者，即效应物半胱天冬酶（casepase- 3和半胱天冬酶-7）。 这些相互作用压制了凋亡命令，从而促进了细胞存活。 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中相互作用所需的结构域。 我们将解决Fas螯合的Met和它的贡献，肝细胞存活，肝脏发育，肝稳态和转化转基因和敲除小鼠模型的生物学相关性。 在目的2中，我们将检验Met是细胞凋亡期间效应物半胱天冬酶（例如半胱天冬酶-3）的破坏作用的关键靶标的假设。 这一假设是基于我们的发现，即Met对细胞存活是必不可少的，并且因为在Met的酪氨酸激酶激活结构域中存在完美的半胱天冬酶切割位点。 重要的是，在这个酪氨酸激酶激活域（在假定的半胱天冬酶裂解位点，我们已经确定）的种系和零星突变已被报道。 我们将研究这样的假设，即这些突变使Met对胱天蛋白酶切割不敏感，从而导致细胞对凋亡产生抗性并导致恶性转化。 在目标3中，我们提出了一种假设，即Met蛋白已经进化出一种新的机制来逃避/抑制凋亡途径的执行者，即效应器半胱天冬酶。 我们提出，通过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实现的。 在该区域，我们发现了八肽序列DNADDEVD。 由于该肽序列具有串联的完美效应物半胱天冬酶切割位点（即半胱天冬酶-3），我们提出在细胞凋亡期间，该肽序列被这些半胱天冬酶切割，导致形成四肽DEVD，一种众所周知的半胱天冬酶-3和半胱天冬酶-7的有效抑制剂。 在目标4中，我们将检验这样的假设：met基因是在细胞应激时被诱导的，以保护细胞免受凋亡，并且是NF-kappaB上调的靶点，NF-kappaB是一种已知对胚胎发育期间肝细胞生存至关重要的转录因子。

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.