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）：由于细胞死亡的一种生化不同形式的凋亡是通过化学疗法或激素操纵引发的癌细胞中主要反应之一，因此对了解凋亡过程的调节具有很大的兴趣。在过去的二十年中进行的研究表明，BCL-2家族成员在细胞生命/死亡决策中起关键作用。特别是，人们认为tat bax和bak会导致线粒体外膜通透性（MOMP）；抗凋亡家庭成员，例如Bcl-2抑制MOMP；通过与Bax/Bak（“直接激活剂”）结合或结合抗催化剂（“敏化器”），仅BH3的成员促进了MOMP。如何激活Bax和Bak以及如何引起MOMP的理解。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，我们首次表明bak寡聚是一个两步过程涉及BH3蛋白*BAK异二聚体的初始形成，然后在BAK HOMO-OLIGOMERS形成过程中释放仅BH3蛋白。我们还绘制了仅BH3蛋白质和BAK上的相互作用域，并在细胞环境中确认了它们在BAK激活中的参与。重要的是，该分析的结果表明BAK寡聚化与当前BAX激活模型之间存在实质性差异。此外，我们的结果暗示NOXA，例如BIM和TBID，是仅激活剂BH3蛋白。为了建立我们的结果，我们现在建议1）研究仅BH3肽对BAK单体的影响，从而确认了假定的结合位点，并提供了对为寡聚化准备BAK的结构变化的见解； 2）阐明巴克的结构通过形态学，生化和计算方法的结合，旨在提供有关BAK的作用和MOMP过程的新信息； 3）检查NOXA的翻译后调节，我们已将其确定为关键参与者硼替佐米诱导的淋巴样细胞凋亡过程中的BAK激活。如果成功的话，提出的实验不仅将为BAK激活过程提供新的见解，还将产生有关日益广泛使用的抗淋巴瘤剂的作用的重要新信息。公共卫生相关性：在2010年诊断出患有癌症的140万美国人中，很大一部分将接受化学疗法和/或荷尔蒙治疗。成功的情况下，这种疗法的主要结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中起关键作用。特别是，蛋白质的bax和bak被认为是通过在称为外部线粒体外膜的细胞内边界中打孔来触发死亡过程的，从而激活了生化变化，最终导致细胞凋亡。尚不完全了解Bax和Bak的激活方式，以及它们随后如何透化膜。在早期观察结果的基础上，我们最近开发了一种使用高度纯化的蛋白来研究BAK激活和膜通透性的方法。现在，我们建议表征活化的BAK分子，研究BAK激活过程，并探索NOXA的化学疗法调节调节，NOXA是BAK激活剂之一。如果成功，则提出的实验将为激活BAK并进入硼替佐米的作用的多种抗癌药物的作用提供新的机械洞察力。