Anticancer drug-induced BH3-only protein.Bak interactions

抗癌药物诱导的 BH3-only 蛋白.Bak 相互作用

• 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
• 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' 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

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）：由于细胞凋亡是细胞死亡的一种生物化学上不同的形式，是化疗或激素操纵在癌细胞中触发的主要反应之一，因此对理解细胞凋亡过程的调节有很大的兴趣。过去二十年的研究表明，Bcl-2家族成员在细胞的生死决定中起着关键作用。特别地，达特Bax和巴克引起线粒体外膜透化（MOMP）;抗凋亡家族成员如Bcl-2抑制MOMP;并且仅BH 3亚家族的成员通过结合Bax/巴克（“直接激活剂”）或结合抗凋亡剂（“敏化剂”）促进MOMP。Bax和巴克是如何被激活的以及它们是如何引起MOMP的还不完全清楚。在初步研究中，我们已经检查了BH 3-唯一的蛋白质介导的巴克激活。使用纯化的蛋白质，我们首次证明了巴克寡聚化是一个重要的过程。 两步过程，包括最初形成BH 3蛋白 * 巴克异二聚体，然后在形成巴克同源寡聚体期间释放仅BH 3蛋白。我们还绘制了仅BH 3蛋白和巴克上的相互作用结构域，并确认了它们在细胞环境中参与了巴克激活过程。重要的是，该分析的结果表明巴克寡聚化和Bax激活的每个当前模型之间的实质性差异。此外，我们的研究结果暗示Noxa，像Bim和tBid一样，是一种仅激活BH 3的蛋白质。为了建立我们的结果，我们现在建议1）检查仅BH 3肽结合在巴克单体上的效果，从而确认推定的结合位点并提供对使巴克准备用于寡聚化的结构变化的洞察; 2）阐明巴克的结构 通过形态学，生物化学和计算方法的组合，旨在提供关于巴克的作用和MOMP过程的新信息;和3）检查Noxa的翻译后调节，我们已经确定为MOMP的关键参与者。 硼替佐米诱导淋巴细胞凋亡过程中的巴克激活。如果成功，所提出的实验将不仅提供新的见解的过程中的巴克激活，但也产生重要的新信息的作用越来越广泛使用的抗淋巴瘤剂。 公共卫生相关性：在2010年被诊断患有癌症的140万美国人中，有相当一部分人将接受化疗和/或激素治疗。当成功时，这种疗法的主要结果之一是在易感癌细胞中诱导凋亡。一组称为Bcl-2家族成员的蛋白质在决定细胞是否发生凋亡方面起着关键作用。特别是，蛋白Bax和巴克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，从而激活最终导致细胞凋亡的生化变化。Bax和巴克是如何被激活的，以及它们随后是如何使膜透化的，还不完全清楚。在早期观察的基础上，我们最近开发了一种使用高度纯化的蛋白质研究巴克活化和膜透化的方法。我们现在建议表征活化的巴克分子，研究巴克活化过程，并探索Noxa（巴克活化剂之一）的化疗调节。如果成功，拟议的实验将提供新的机制洞察各种抗癌药物的行动，激活巴克和硼替佐米的行动。