Dissecting and Targeting Deregulated Mitochondrial Apoptosis in Human Cancer

 描述(申请人提供)：bcl2家族的凋亡蛋白调节细胞的生命和死亡之间的关键平衡。这一重要信号转导网络的解除调控导致了多种人类癌症的发生、维持和化疗耐药。我的实验室正在解决的问题是，在体内平衡和恶性转化过程中，bcl2家族蛋白之间的相互作用如何调节它们的结构变化和信号功能。由于bcl2蛋白的相互作用决定了细胞在应激反应中的生存或死亡，我们相信这些结构-功能研究将为基本的生物学机制和药物调节它们的机会提供更多的信息。三十年来对bcl2家族的研究已经导致了第一种小分子药物在bcl2依赖的癌症中引起反应性细胞凋亡。尽管取得了这一显著的科学进步，并重新承诺靶向癌症中的bcl2蛋白，但关于这个复杂的蛋白家族如何调控线粒体凋亡的基本问题仍然存在。事实上，它们主要的膜定位使bcl2家族蛋白的研究具有极大的挑战性。BAX和BAK的激活形式如何自组装成有毒的寡聚体毛孔--正是介导细胞凋亡的死亡通道--尚不清楚。抗凋亡蛋白如bcl2是如何改变它们在膜内的结构以阻断bax/BAK激活的各个步骤的，目前尚不清楚。事实上，bcl2蛋白的许多保守结构域之间的接触表面的全谱是未知的。也许最令人困惑的问题是，这些结构相似的蛋白质如何具有截然相反的功能，无论是作为细胞脱氧核糖核酸的抑制物还是激活剂，基本上仍然未知。我们通过广泛的实验方法解决这些机械问题，跨越化学、结构生物学、蛋白质组学、生物化学、细胞生物学、小鼠建模和药理学。例如，我们开发了新的化学工具来解剖和靶向bcl2家族蛋白相互作用，提出了利用光反应结构肽和质谱学快速准确地确定相互作用位点的新方法，目前正在应用氢-氢交换质谱仪首次实时研究bcl2家族在膜上的构象变化。我们的R35癌症研究计划的目标包括确定bax和bak的构象激活和同源寡聚机制(S)，表征抗凋亡bcl2蛋白的BH4结构域抑制bax和bak的新机制，以及研究控制bcl2蛋白凋亡功能的新的变构机制。在每一种情况下，都将利用结构-功能的洞察力来开发针对癌症中的凋亡抵抗的新方法。作为一名化学生物学家和执业的儿科肿瘤学家，我一直致力于破译bcl2家族介导的癌症机制，以便对其蛋白质相互作用生物学的新见解能够为下一代人类癌症的促凋亡疗法提供信息。

 DESCRIPTION (provided by applicant): The BCL-2 family of apoptotic proteins regulates the critical balance between cellular life and death. Deregulation of this essential signal transductio network drives the development, maintenance, and chemoresistance of a broad spectrum of human cancers. My laboratory is addressing the questions of just how interactions among BCL-2 family proteins regulate their structural changes and signaling functions during homeostasis and malignant transformation. Because BCL-2 protein interactions determine whether the cell will live or die in response to stress, we believe these structure-function studies will both infor basic biological mechanisms and opportunities to pharmacologically modulate them. Thirty years of BCL-2 family research has led to the first small molecule drug to reactive apoptosis in BCL-2 dependent cancer. Despite this remarkable scientific progress and the renewed promise of targeting BCL-2 proteins in cancer, fundamental questions remain about how this complex protein family regulates mitochondrial apoptosis. Indeed, their predominant membrane localization has made BCL-2 family proteins remarkably challenging to study. How the activated forms of BAX and BAK self-assemble into toxic oligomeric pores - the very death channels that mediate apoptosis - is unknown. How anti-apoptotic proteins such as BCL-2 change their structure within the membrane to block the various steps of BAX/BAK activation is unknown. Indeed, the full spectrum of contact surfaces between the many conserved domains of BCL-2 proteins is unknown. Perhaps the most perplexing question of how such structurally similar proteins can have diametrically opposite functions, as either inhibitors or activators of cell deat, remains essentially unknown. We address these mechanistic questions with broad experimental approaches, spanning chemistry, structural biology, proteomics, biochemistry, cell biology, mouse modeling, and pharmacology. For example, we have developed new chemical tools to dissect and target BCL-2 family protein interactions, advanced new methods to rapidly and accurately identify interaction sites using photoreactive structured peptides and mass spectrometry, and are currently applying hydrogen- deuterium exchange mass spectrometry to study BCL-2 family conformational changes in the membrane in real-time for the first time. Our goals for this R35 cancer research program include defining the conformational activation and homo-oligomerization mechanism(s) of BAX and BAK, characterizing a novel mechanism for BAX and BAK suppression by the BH4 domains of anti-apoptotic BCL-2 proteins, and investigating a new allosteric mechanism that controls the apoptotic functionalities of BCL-2 proteins. In each case, the structure- function insights will be harnessed to develop new approaches for targeting apoptotic resistance in cancer. As a chemical biologist and practicing pediatric oncologist, I have dedicated my research laboratory to deciphering BCL-2 family-mediated cancer mechanisms so that fresh insights into their protein interaction biology can inform the next generation of pro-apoptotic therapies for human cancer.