The Mitochondrial Permeabilty Transition in Apoptosis

细胞凋亡中的线粒体通透性转变

• 批准号: 6607261
• 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/6607261
• 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.

已有研究表明，线粒体通过释放促凋亡因子在调节细胞凋亡的内在途径中发挥重要作用。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。在细胞凋亡的过程中，线粒体的生理发生了几个变化，其中包括早期的线粒体pH变化。在细胞凋亡过程中，线粒体生理发生的许多变化被认为是由于线粒体通透性转变(MPT)孔的打开而发生的。MPT孔被认为(至少)由外膜电压依赖性阴离子通道(VDAC)、内膜腺核苷酸转位酶(ANT)和基质亲环素D(CypD)组成。Bcl2蛋白家族的成员调控MPT孔的活性，可能是通过在线粒体外膜上直接形成孔和/或通过它们之间或与MPT孔的成分相互作用来调节的。然而，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和与Bcl2家族成员相互作用，目前尚不清楚。我们实验室和Reed及同事的最新证据表明，Bcl-2家族成员调控细胞凋亡诱导的线粒体pH变化，并提示线粒体pH可能是调控细胞凋亡过程中MPT孔活动的机制之一。因此，我们的具体目标是：1)确定MPT孔成分在原位凋亡过程中是否以一种pH依赖的方式相互作用。我们将使用我们最近开发的双融合FRET技术，确定这些蛋白质在凋亡之前或期间是否存在原位相互作用，以及线粒体ph值是否调节这些相互作用。2.确定抗凋亡的Bcl2(以及促凋亡的Bax样蛋白)家族成员发挥生物活性的机制。我们将调查Bcl2家族成员的造孔活动和/或他们与MPT孔的特定成分相互作用的能力是否在他们的作用机制中起重要作用(S)。在拟议的研究中，我们将检验以下假设：1)线粒体pH的变化调节MPT孔的打开和细胞色素c的释放；2)MPT孔的打开需要Bax与ANT的相互作用；3)促凋亡和抗凋亡的Bcl-2家族成员通过与VDAC的相互作用来调节MPT孔的活性；4)促凋亡和抗凋亡的Bcl-2家族成员通过直接通过其成孔活动或通过与其他家族成员或MPT孔的成分相互作用间接地调节线粒体的pH来发挥其凋亡调节活性；5)线粒体含有诱导MPT孔所需的依赖于pH的半胱氨酸酶活性。这些研究既有意义又有创新，因为它们涉及一个基本的生物过程--细胞凋亡的机制，涉及生物体的一些正常和病理活动，并使用新的和新的方法来获得其他实验策略无法实现的信息。