Role of Bax and pH in Death by Cytokine Withdrawal

描述(申请人提供)：调节细胞凋亡的基因产物在癌症中经常发生突变。正常情况下，细胞凋亡性死亡是淋巴细胞发育和维持的重要部分，细胞因子等生存或营养信号的丧失是导致死亡的主要原因。因此，阐明新的调节蛋白和细胞因子撤除过程中的代谢变化对于理解细胞死亡过程至关重要，更重要的是发现在癌变过程中发生突变的新成分。为此，细胞因子撤除诱导细胞因子依赖细胞系凋亡的研究已被证明是成功的。在最初的研究中，细胞因子信号的丢失导致细胞质快速和瞬时的碱化，这是由pH调节蛋白、钠/氢交换器1(NHE1)和p38MAPK(MAPK)介导的。这种pH的升高被认为是导致促凋亡蛋白Bax的线粒体易位，从而导致细胞死亡。作为第一个目标，我们提出了一个由两部分组成的研究，以严格检验碱性pH通过中和末端的带电氨基酸而导致Bax向线粒体移动的假设。首先，将评估与pH无关的修饰或对Bax的间接pH影响。其次，将制造一系列Bax突变体，以定位建议的pH敏感部位，并测试在凋亡和非凋亡条件下的线粒体易位。预期的结果是证明Bax是一种对pH有反应的蛋白质。在第二个目标中，将评估pH升高的中介物NHE1在细胞凋亡中的作用。以前的工作确定了NHE1上的四个候选位点作为p38MAPK磷酸化的靶点。这些位点的重要性将首先通过使用突变的GST-NHE1融合蛋白作为磷酸化底物进行p38MAPK激酶检测，以及第二，在NHE1缺失的细胞系中在凋亡和非凋亡条件下表达全长NHE1突变蛋白来确定。预计表达突变型NHE1的细胞不会碱化，Bax不会移位，死亡将被抑制。未来的目标包括通过NHE分析碱化机制和阐明Bax在线粒体损伤中的作用。这项工作将是一个学术研究项目的基础，作为一个终身教职跟踪的教师，从独特的pH介导的细胞凋亡变化的角度来识别和应用在癌症中突变的新的凋亡因子。

DESCRIPTION (provided by applicant): Gene products that regulate apoptosis are frequently mutated in cancer. Normally, apoptotic cell death is an essential part of lymphocyte development and maintenance, with the loss of a survival or trophic signal, such as a cytokine, as principal trigger for death. Hence, elucidation of novel regulatory proteins and metabolic changes during cytokine withdrawal is of vital importance for the understanding of the cell death process and more so for discovering new components mutated during carcinogenesis. To this end, the study of apoptosis induced by cytokine withdrawal in cytokine-dependent cell lines has proven successful. In initial studies, loss of cytokine signaling led to a rapid and transient alkalinization of the cytosol, mediated by the pH regulating protein, the sodium/hydrogen exchanger1 (NHE1) and p38 MAP kinase (MAPK). This rise in pH is proposed to cause the mitochondrial translocation of the pro-apoptotic protein, Bax, resulting in cell death. As the first aim, a two-part study is proposed to rigorously test the hypothesis that alkaline pH causes the movement of Bax to mitochondria by neutralizing charged amino acids in the termini. First, pH-independent modifications or indirect pH effects on Bax will be evaluated. Second, a series of Bax mutants will be made to target proposed pH sensitive sites and tested for mitochondrial translocation under apoptotic and non-apoptotic conditions. The expected outcome is to prove that Bax is a pH-responsive protein. In the second aim, the mediator of the pH rise, NHE1, will be evaluated for function during apoptosis. Previous work identified four candidate sites on NHE1 as targets for p38 MAPK phosphorylation. The importance of these sites will be determined by, first, performing p38 MAPK kinase assays using mutated GST-NHE1 fusion proteins as substrates for phosphorylation, and second, expressing full-length NHE1 mutant proteins in an NHE1-deficient cell line under apoptotic and non-apoptotic conditions. It is expected that mutant NHE1 expressing cells will not alkalinize, Bax will not translocate and death will be inhibited. Future aims include analysis of the alkalinization mechanism through NHE and elaboration of the role of Bax in mitochondrial damage. This work will be the foundation of an academic research program, as a tenure-tracked faculty, for the identification and application of novel apoptotic factors mutated in cancer from the unique perspective of pH-mediated apoptotic changes.