Structure-Function Analysis of Pro-Apoptotic BMRP and Its Role as a Tumor Suppressor

摘要 细胞的死亡是后生动物正常发育所必需的，也是 维持组织的动态平衡和成年生物体的正常功能。异常的水平 细胞凋亡与人类疾病的病因学有关，包括癌症、 免疫系统、不孕不育、神经退行性疾病、心脏骤停和中风损害。在这种情况下 在癌症中，细胞凋亡受损是抵抗化疗和放射治疗的主要原因。BCL-2是一种抗肿瘤药物 在这一细胞死亡过程中起主要调节作用的凋亡蛋白，尽管 在调查中，对其作用机制和监管的几个方面仍然不太了解。至 进一步了解Bcl2‘S抗细胞凋亡作用的分子机制及其机制 BMRP是一种新的Bcl-2相互作用蛋白 结合蛋白。目前的知识表明，BMRP是一种促凋亡蛋白，在肿瘤- 抑制者角色。我们的长期目标是加强我们对分子机制的理解 介导Bc l-2‘S促生存活性及其调控，并利用这一知识为肿瘤的发展提供依据 新的治疗方法。我们的假设是BMRP是分子中的一个相关成分 凋亡机制，它的促凋亡活性抵消了Bcl-2的活性，这一机制 研究将为调控细胞凋亡提供新的靶点和工具。增加了对分子的了解 BMRP和Bc l-2功能及其调控的机制基础将加深我们对BMRP和Bc l-2的认识 在细胞凋亡途径中的作用和信号转导，这将有助于新药的开发 预防和/或治疗涉及非调控细胞凋亡的人类疾病。提出了两个具体目标，以 检验我们的假设。在第一个目标中，将产生BMRP的嵌合突变体和丙氨酸替代突变体 也为未来新的肿瘤联合疗法的开发确定一个或多个杀伤性多肽 作为其促凋亡活性所必需的氨基酸残基。此外，这些突变体结合的能力 用酵母双杂交法和免疫共沉淀法检测TO-Bcl2。第二个目标将是 通过测定BMRP的表达水平来研究BMRP作为肿瘤抑制因子的作用 肿瘤细胞中蛋白质和信使核糖核酸的表达，并分析异常的遗传机制 BMRP在这些癌细胞系中的表达。拟议的研究是创新的，因为它将建立 BMRP功能和调控的未知分子方面，以及BMRP作为肿瘤抑制因子的作用。这 知识与公共卫生有关，因为它应该为设计 治疗和/或预防人类疾病的高级疗法，其特点是放松管制 细胞凋亡，如癌症。

Abstract Cell death by apoptosis is required for the normal development of metazoans, as well as for the maintenance of tissue homeostasis and normal functioning of the adult organism. Abnormal levels of apoptosis have been implicated in the etiology of human diseases including cancer, disorders of the immune system, infertility, neurodegenerative diseases, and cardiac arrest and stroke damage. In the case of cancer, impaired apoptosis is a major cause of resistance to chemo- and radio-therapies. Bcl-2 is an anti- apoptotic protein that plays a major regulatory role in this process of cell demise and, despite intense investigation, several aspects of its mechanisms of action and regulation are still not well understood. To gain further insight into the molecular mechanisms that mediate Bcl-2's anti-apoptotic function and its regulation, we conducted a search of Bcl-2 interacting proteins, which identified BMRP as a novel Bcl-2 binding protein. Current knowledge suggests that BMRP is a pro-apoptotic protein that plays a tumor- suppressor role. Our long-term goal is to enhance our understanding of the molecular mechanisms that mediate Bcl-2's pro-survival activity and its regulation, and to exploit this knowledge for the development of novel therapeutic approaches. Our hypothesis is that BMRP is a relevant component of the molecular apoptotic machinery, that its pro-apoptotic activity counteracts the activity of Bcl-2, and that mechanistic studies will yield novel targets and tools for modulating apoptosis. An increased knowledge of the molecular mechanistic basis of BMRP and Bcl-2 function and modulation will enhance our understanding of the players and signaling in apoptosis pathways, which will contribute to the development of novel drugs to prevent and/or treat human diseases involving deregulated apoptosis. Two specific aims are proposed to test our hypothesis. In the first aim, chimeric and alanine substitution mutants of BMRP will be generated to identify a killer peptide (or peptides) for future development of novel tumor combinatorial therapies, as well as amino acid residues essential to its pro-apoptotic activity. Additionally, the ability of these mutants to bind to Bcl-2 will be tested by yeast Two-Hybrid and co-immunoprecipitation assays. A second aim will investigate the role of BMRP as a tumor suppressor by determining the expression levels of the BMRP protein and mRNA in cancer cell lines, and analyzing the genetic mechanisms responsible for abnormal BMRP expression in these cancer cell lines. The proposed research is innovative, as it will establish unknown molecular aspects of BMRP function and regulation, and BMRP's role as a tumor suppressor. This knowledge is relevant to public health, since it should provide novel targets and tools for the design of advanced therapies for the treatment and/or prevention of human diseases characterized by deregulated apoptosis, such as cancer.