The Mitochondrial Permeabilty Transition in Apoptosis

细胞凋亡中的线粒体通透性转变

• 批准号: 6763185
• 负责人: BRIAN A. HERMAN
• 金额: $ 25.29万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-15 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6763185
• 关键词: BCL2 gene /protein; Bax gene /protein; acidity /alkalinity; adenine nucleotides; apoptosis; confocal scanning microscopy; cysteine endopeptidases; cytochrome c; fluorescence resonance energy transfer; membrane channels; membrane permeability; membrane transport proteins; mitochondria; mitochondrial membrane; peptidylprolyl isomerase; protein protein interaction; voltage gated channel

mitochondria have been shown to play a critical role in the regulation of the intrinsic pathway to apoptosis through the release of apoptogenic factors 9i.e. cytochrome c) that activate specific class of cysteine proteases (caspases), leading to cell death. During apoptosis, several alterations to mitochondria physiology occur, amongst which is an early change in mitochondrial pH. Many of the changes in mitochondria physiology that occur during apoptosis are thought to take place because of the opening of a pore, know as the mitochondrial permeability transition (MPT) pore. The MPT pore is hypothesized to consist (minimally) of the outer membrane voltage-dependent anion channel (VDAC), the inner membrane adenine nucleotide translocase (ANT) and matrix cyclophilin D (CypD). Members of the Bcl-2 family of proteins modulate the activity of the MPT pore, potentially via direct formation of pores in the outer mitochondrial membrane and/or through interaction amongst themselves or with components of the MPT pore. However, the functional and structural components that constitute the MPT pore in situ, their regulation, and how they interact with each other and Bcl-2 family members, is not known. Recent evidence from our laboratory and those of Reed and colleagues, indicate that Bcl-2 family members modulate apoptosis-induced alterations in mitochondrial pH, and suggest that mitochondrial pH may one of the mechanisms regulating MPT pore activity during apoptosis. Therefore, our specific aims are: 1) Determine whether the MPT pore components interact in a pH-dependent fashion during apoptosis in situ. We will determine whether these proteins interact in situ before or during apoptosis, and if mitochondrial ph regulates these interactions, using our recently developed "two-fusion" FRET technique. 2. Determine the mechanisms by which the anti-apoptotic Bcl-2 (and conversely the proapoptotic Bax-like) family members exert their biological activities. We will investigate whether the pore-forming activities of Bcl-2 family members and/or their ability to interact with specific components of the MPT pore are important in their mechanism(s) of action. In the proposed studies we will test the following hypotheses: 1)Alterations in mitochondrial pH regulate the opening of the MPT pore and cytochrome c release during apoptosis; 2) Bax interaction with the ANT is required for opening of the MPT pore; 3)Pro-and anti-apoptotic Bcl-2 family members modulate MPT pore activity via interactions with the VDAC; 4) Pro- and anti-apoptotic Bcl-2 family members exert their apoptotic regulatory activity by modulating mitochondrial pH, either directly through their pore forming activity or by indirectly by interacting with other family members or with constituents of the MPT pore; and 5) mitochondria contain pH-dependent caspases activity that is required for induction of the MPT pore. These studies are both significant and innovation, as they address the mechanisms of a fundamental biological process, apoptosis, involved in a number of normal and pathological activities of organisms and employ new and novel methods to obtain information that cannot be achieved with other experimental strategies.

线粒体已显示出通过释放凋亡因子9i.e的释放在凋亡的内在途径中起关键作用。细胞色素c）激活特定类别的半胱氨酸蛋白酶（caspase），导致细胞死亡。 在凋亡过程中，线粒体生理发生了几种改变，其中是线粒体pH的早期变化。 凋亡过程中发生的线粒体生理学的许多变化都被认为是由于孔口的打开而发生的，被称为线粒体通透性转变（MPT）孔。 假设MPT孔（最小）外膜电压依赖性阴离子通道（VDAC），内膜腺嘌呤核苷酸易位酶（ANT）和基质环蛋白D（CYPD）组成。 Bcl-2蛋白质家族的成员可以通过直接形成线粒体外膜中的孔和/或通过自身的相互作用或与MPT孔的成分进行相互作用来调节MPT孔的活性。但是，尚不清楚构成原位MPT孔的功能和结构成分，它们的调节以及它们如何相互作用以及BCL-2家族成员的相互作用。 我们的实验室和REED及其同事的证据表明，Bcl-2家族成员调节细胞凋亡诱导的线粒体pH的改变，并表明线粒体pH值可能是调节凋亡过程中MPT孔活性的机制之一。 因此，我们的具体目的是：1）确定在原位凋亡期间，MPT孔成分是否以pH依赖性方式相互作用。 我们将使用我们最近开发的“两种融合” FRET技术来确定这些蛋白在凋亡之前或凋亡期间是否在原位相互作用。 2。确定抗凋亡BCl-2（相反，类似于Bax的抗凋亡Bcl-2）的家族成员发挥其生物学活性的机制。 我们将研究Bcl-2家族成员的孔形成活性和/或与MPT孔特定成分相互作用的能力是否在其作用机制中很重要。在拟议的研究中，我们将检验以下假设：1）线粒体pH的改变调节凋亡过程中MPT孔隙和细胞色素C释放的打开； 2）与ANT的Bax相互作用是开放MPT孔需要的； 3）亲和抗凋亡的Bcl-2家族成员通过与VDAC的相互作用调节MPT孔的活性； 4）促和抗凋亡的Bcl-2家族成员通过调节线粒体pH，直接通过其孔形成活性或与其他家庭成员相互作用或与MPT孔的成分进行调节，从而发挥其凋亡调节活性； 5）线粒体含有pH依赖性的caspases活性，这是诱导MPT孔所需要的。 这些研究既重要又是创新的，因为它们探讨了一种基本生物学过程的机制，凋亡涉及生物体的许多正常和病理活性，并采用了新的和新颖的方法来获得其他实验策略无法实现的信息。