Novel Proapoptotic BH3-only Proteins in Cancer Apoptosis

描述（申请人提供）：多种癌细胞在凋亡途径上存在缺陷。Bcl-2蛋白家族成员在调节细胞凋亡中起关键作用，并拥有4个保守的Bcl-2同源结构域（BH1、BH2、BH3和BH4）中的至少1个。BH3结构域是促凋亡“仅BH3”分子中唯一存在的结构域(1)，通过调节Bcl-2家族成员的二聚化和多聚化，在蛋白-蛋白相互作用和细胞凋亡中发挥重要作用。迄今为止，大约有21个人类基因被发现编码Bcl-2家族的蛋白质，其中14个是仅bh3的促凋亡蛋白。这表明BH3-only蛋白在不同类型细胞凋亡中具有重叠、互补和/或特异性作用。因此，本研究的总体目标是从人类蛋白质组中鉴定和表征新的BH3-only蛋白，并描述新鉴定的蛋白在癌细胞死亡中的功能。我们假设在人类基因组中还有其他基因编码BH3-only蛋白，这些蛋白与Bcl-2家族的其他成员和/或其他分子相互作用，以细胞类型和/或信号特异性方式调节细胞凋亡。我们进一步假设，一些新发现的BH3-only蛋白在正常生理中具有额外的（即非凋亡）功能。在初步研究中，我们利用生物信息学数据挖掘方法鉴定了3种新的BH3-only蛋白。候选基因为载脂蛋白L6 （ApoL6）、ApoL。1和BH3- Only Member B (BOMB)，克隆并在肿瘤模型中功能表达。通过瞬时表达分析，我们发现ApoL6、ApoL1和BOMB促进了p53缺失的HCT116细胞的细胞死亡。此外，通过在另一种p53缺失的癌症模型DLD-1中构建和表征可诱导的ApoL6基因，我们发现ApoL6的表达可诱导DLD-1细胞线粒体介导的凋亡。这些假设将通过5个具体目标得到进一步验证。揭示新的BH3-only蛋白及其相互作用分子的功能，有助于提高我们对BH3-only蛋白和脂质介导的细胞凋亡在癌症治疗中的理解和潜在利用。

DESCRIPTION (provided by applicant): Many kinds of cancer cells possess defects in the pathways of apoptosis. Members of the Bcl-2 family of proteins play pivotal roles in regulating apoptosis and possess at least 1 of 4 conserved Bcl-2-Homology (BH) domains, designated, BH1, BH2, BH3, and BH4. The BH3 domain is the only one (1) present in pro-apoptotic "BH3-only" molecules and plays important roles in protein-protein interactions and apoptosis by regulating dimerization and multimerization of the Bcl-2 family members. To date, approximately 21 human genes have been found that encode proteins belonging to the Bcl-2 family, 14 of which are BH3-only pro-apoptotic proteins. This shows that the BH3-only proteins are important and possess overlapping, complementary and/or specific roles in different types of cells in apoptosis. Therefore, the overall goals of this study are to identify and characterize novel BH3-only proteins from the human proteome and to delineate functions of the newly identified proteins in cancer cell death. We hypothesized that there are additional genes encoding BH3-only proteins in the human genome that interact with other members of the Bcl-2 family and/or other molecules in regulating apoptosis in cell type and/or signaling-specific manners. We further hypothesize that some of the newly identified BH3-only proteins possess additional (i.e., non-apoptogenic) functions in normal physiology. In the preliminary studies, we utilized the bioinformatic data-mining approach to identify 3 novel BH3-only proteins. The candidate genes, apolipoprotein L6 (ApoL6), ApoL.1 and BH3- Only Member B (BOMB), were cloned and functionally expressed in cancer models. By transient expression analysis, we showed that ApoL6, ApoL1 and BOMB promoted cell death in p53-null HCT116 cells. Furthermore, by constructing and characterizing an inducible ApoL6 gene in another p53-null cancer model, DLD-1, we found that expression of ApoL6 induced a mitochondria-mediated apoptosis in DLD-1 cells. These hypotheses will be tested further with 5 specific aims. Deciphering functions of the novel BH3-only proteins and their interacting molecules should enhance our understanding, and potential utilization, of BH3-only proteins and lipid-mediated apoptosis in treatment of cancer.