Structural, biochemical and genetic analysis of the newly discovered Apaf-1-binding protein CABY

细胞凋亡的抑制被认为是肿瘤发生的主要原因，并且鉴定进一步的细胞凋亡调节蛋白作为分子治疗的潜在靶点已成为重要目标。在线粒体凋亡途径的激活过程中，组装了大的胞质溶酶体复合物，其由寡聚衔接蛋白Apaf-1、细胞色素c、dATP/ATP和Caspase-9组成。凋亡体的形成导致效应物半胱天冬酶-3的进一步募集和活化，随后导致细胞死亡。这种途径会被大量刺激激活，包括癌症治疗期间的放疗和化疗。我们最近发现了一种新的Apaf-1结合蛋白，我们将其命名为CABY，它使细胞对激活线粒体凋亡途径的死亡诱导刺激敏感。在拟议的项目中，我们计划对CABY进行详细的分子和结构分析。为此，我们将结合联合收割机的生化方法与核磁共振光谱，以揭示精确的分子机制，通过CABY影响激活的复合体。使用条件性CABY基因敲除小鼠模型的遗传学研究将使我们能够研究CABY的生理功能。促凋亡蛋白可以作为肿瘤抑制分子发挥作用。我们之前的结果表明，CABY在胃肠道间质瘤（GIST）中下调，我们将进行表达研究以查明CABY活性是否在其他肿瘤类型中减弱。总的来说，这些实验将有助于提高对细胞凋亡调控的理解，并有助于开发癌症和癌症抗性的新治疗方法。

Inhibition of apoptosis is recognized as a major cause of tumorigenesis, and identification of further apoptosis-regulating proteins as potential targets for molecular therapy has become an important goal. During activation of the mitochondrial apoptosis pathway, the large cytosolic apoptosome complex is assembled, which consists of the oligomerized adaptor protein Apaf-1, Cytochrome c, dATP/ATP and Caspase-9. Apoptosome formation leads to the further recruitment and activation of the effector Caspase-3 and, subsequently, to cell death. This pathway is activated by a plethora of stimuli, including radiation and chemotherapy during cancer treatment. We recently discovered a novel Apaf-1-binding protein, which we named CABY and which sensitizes cells to death-inducing stimuli activating the mitochondrial apoptosis pathway. In the proposed project we plan to carry out a detailed molecular and structural analysis of CABY. For this purpose, we will combine biochemical approaches with NMR spectroscopy to uncover the precise molecular mechanism through which CABY influences activation of the apoptosome complex. Genetic studies using a conditional CABY knockout mouse model will allow us to investigate the physiological function of CABY. Pro-apoptotic proteins may function as tumor suppressor molecules. Our previous results have shown that CABY is downregulated in gastrointestinal stromal tumors (GISTs), and we will perform expression studies to find out whether CABY activity is diminished in other tumor types. Collectively, the experiments will contribute to an improved understanding of apoptosis regulation and the development of new therapeutic approaches for cancer and cancer resistance.