Anticancer drug-induced BH3-only protein.Bak interactions

抗癌药物诱导的 BH3-only 蛋白.Bak 相互作用

基本信息

批准号：
8273913
负责人：
SCOTT H KAUFMANN
金额：
$ 33.26万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-16 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8273913
关键词：
1-Phosphatidylinositol 3-Kinase American Androgen Antagonists Antineoplastic Agents Apoptosis Apoptotic Area BH3 Domain BH3 peptide Bax protein Binding Binding Sites Biochemical Bortezomib Cell Death Cells Cervix Uteri Cessation of life Colon DNA Damage Development Diagnosis Event Family member Funding Glucocorticoids Head and neck structure Hodgkin Disease Homo Hormonal Induction of Apoptosis Kidney Lead Life Ligands Lung Lymphoid Cell Lymphoma Lysine MEKs Malignant Neoplasms Mantle Cell Lymphoma Maps Mediating Membrane Messenger RNA Methods Modeling Molecular Molecular Conformation Mutation N-terminal NMR Spectroscopy Non-Hodgkin&apos s Lymphoma Noxae Outer Mitochondrial Membrane Participant Peptides Play Positioning Attribute Post-Translational Regulation Process Proteasome Inhibitor Protein Binding Proteins Ptosis Reaction Regulation Resolution Role Site-Directed Mutagenesis Structure Testing Time Up-Regulation anticancer treatment apoptosis in lymphoid cells bak protein cancer cell chemotherapeutic agent chemotherapy cytotoxicity design effective therapy hormone therapy inhibitor/antagonist insight interest killings malignant breast neoplasm member monomer mutant research study resistance mechanism response therapy outcome tumor

项目摘要

DESCRIPTION (provided by applicant): Because apoptosis, a biochemically distinct form of cell death, is one of the major responses triggered in cancer cells by either chemotherapy or hormonal manipulation, there has been substantial interest in understanding regulation of the apoptotic process. Studies performed over the past two decades have demonstrated that Bcl-2 family members play critical roles in cellular life/death decisions. In particular, it is thought tat Bax and Bak cause mitochondrial outer membrane permeabilization (MOMP); antiapoptotic family members such as Bcl-2 inhibit MOMP; and members of the BH3-only subfamily facilitate MOMP by either binding to Bax/Bak ("direct activators") or binding the antiapoptotics ("sensitizers"). How Bax and Bak are activated and how they cause MOMP is incompletely understood. In preliminary studies, we have examined BH3-only protein-mediated Bak activation. Using purified proteins, we have shown for the first time that Bak oligomerization is a two-step process involving initial formation of a BH3 protein*Bak heterodimer followed by release of the BH3-only protein during formation of Bak homo-oligomers. We have also mapped the interacting domains on the BH3-only protein and Bak and have confirmed their involvement during Bak activation in a cellular context. Importantly, the results of this analysis suggest substantial differences between Bak oligomerization and each of the current models of Bax activation. Moreover, our results implicate Noxa, like Bim and tBid, as an activator BH3-only protein. To build on our results, we now propose to 1) examine the effect of BH3-only peptide binding on the Bak monomer, thereby confirming the putative binding site and providing insight into the structural changes that prepare Bak for oligomerization; 2) elucidate the structure of Bak oligomers through a combination of morphological, biochemical and computational approaches designed to provide new information about the action of Bak and the process of MOMP; and 3) examine posttranslational regulation of Noxa, which we have identified as a critical participant in Bak activation during bortezomib-induced apoptosis in lymphoid cells. If successful, the proposed experiments will not only provide new insight into the process of Bak activation, but also yield important new information about action of an increasingly widely used anti-lymphoma agent. PUBLIC HEALTH RELEVANCE: A substantial fraction of the 1.4 million Americans diagnosed with cancer in 2010 will receive chemotherapy and/or hormonal therapy. One of the major outcomes of this therapy, when successful, is the induction of apoptosis in susceptible cancer cells. A group of proteins called Bcl-2 family members play critical roles in determining whether cells undergo apoptosis. In particular, the proteins Bax and Bak are thought to trigger the death process by punching holes in an intracellular boundary called the outer mitochondrial membrane, thereby activating biochemical changes that ultimately lead to apoptosis. How Bax and Bak are activated, and how they subsequently permeabilize membranes, is incompletely understood. Building on earlier observations, we have recently developed a method for studying Bak activation and membrane permeabilization using highly purified proteins. We now propose to characterize the activated Bak molecules, study the Bak activation process, and explore the chemotherapy-modulated regulation of Noxa, one of the Bak activators. If successful, the proposed experiments will provide new mechanistic insight into the action of a variety of anticancer drugs that activate Bak and into the action of bortezomib.

描述（由申请人提供）：由于细胞凋亡（一种生化上独特的细胞死亡形式）是化疗或激素操作在癌细胞中引发的主要反应之一，因此人们对了解细胞凋亡过程的调节产生了浓厚的兴趣。过去二十年进行的研究表明，Bcl-2 家族成员在细胞生/死决策中发挥着关键作用。特别是，人们认为 Bax 和 Bak 会导致线粒体外膜透化 (MOMP)；抗凋亡家族成员如Bcl-2抑制MOMP；仅 BH3 亚家族的成员通过与 Bax/Bak（“直接激活剂”）结合或结合抗凋亡剂（“敏化剂”）来促进 MOMP。 Bax 和 Bak 如何被激活以及它们如何导致 MOMP 尚不完全清楚。在初步研究中，我们检查了仅 BH3 蛋白介导的 Bak 激活。使用纯化的蛋白质，我们首次证明 Bak 寡聚化是一种两步过程，涉及最初形成 BH3 蛋白*Bak 异二聚体，然后在 Bak 同源寡聚体形成过程中释放仅 BH3 蛋白。我们还绘制了 BH3-only 蛋白和 Bak 上的相互作用域，并证实了它们在细胞环境中参与 Bak 激活过程。重要的是，该分析的结果表明 Bak 寡聚化与当前每种 Bax 激活模型之间存在显着差异。此外，我们的结果表明 Noxa 与 Bim 和 tBid 一样，是一种仅激活 BH3 的蛋白。为了以我们的结果为基础，我们现在建议 1) 检查 BH3-only 肽结合对 Bak 单体的影响，从而确认假定的结合位点并深入了解为 Bak 寡聚化做准备的结构变化； 2）阐明Bak的结构通过结合形态学、生物化学和计算方法来制备低聚物，旨在提供有关 Bak 作用和 MOMP 过程的新信息； 3）检查 Noxa 的翻译后调控，我们已将其确定为关键参与者硼替佐米诱导淋巴细胞凋亡期间的 Bak 激活。如果成功，所提出的实验不仅将为 Bak 激活过程提供新的见解，而且还将产生有关日益广泛使用的抗淋巴瘤药物作用的重要新信息。公共健康相关性：2010 年被诊断患有癌症的 140 万美国人中，很大一部分将接受化疗和/或激素治疗。如果成功，这种疗法的主要成果之一是诱导易感癌细胞凋亡。一组称为 Bcl-2 家族成员的蛋白质在确定细胞是否发生凋亡方面发挥着关键作用。特别是，蛋白质 Bax 和 Bak 被认为通过在称为线粒体外膜的细胞内边界上打孔来触发死亡过程，从而激活生化变化，最终导致细胞凋亡。 Bax 和 Bak 如何被激活，以及它们随后如何透化膜，尚不完全清楚。基于早期的观察，我们最近开发了一种使用高度纯化的蛋白质研究 Bak 激活和膜透化的方法。我们现在建议表征激活的 Bak 分子，研究 Bak 激活过程，并探索 Bak 激活剂之一 Noxa 的化疗调节。如果成功，拟议的实验将为激活 Bak 的各种抗癌药物的作用和硼替佐米的作用提供新的机制见解。