Molecular Identity of the Cardiac Mitochondrial Pore

项目总结 线粒体功能障碍是心肌细胞死亡的根本原因，因此在 许多心脏疾病的发展。这种线粒体功能障碍通常是由开放的 线粒体通透性转换(MPT)孔，导致内部线粒体迅速增加 膜通透性。这反过来会导致ATP耗尽，产生活性氧物种，线粒体 肿胀和破裂。因此，我们的长期目标是确定构成MPT孔的蛋白质 并了解调控这种复合体的分子机制。MPT的毛孔最初是 被认为由外膜上的电压依赖性阴离子通道(VDAC)组成，腺嘌呤 内膜中的核苷酸转位酶(ANT)，以及内膜中的调节蛋白亲环素-D(CypD) 矩阵。然而，遗传学研究表明，VDAC和ANT对于MPT来说是必不可少的。因此，CypD仍然 仍然是MPT孔道复合体中唯一真正的成员。重要的是，直接 CypD的调控及其前MPT功能尚待阐明。特别是，作用于CypD的结合蛋白 抑制CypD的功能以及CypD磷酸化在其中所起的额外作用尚未确定 对MPT和细胞死亡的监管尚未得到全面解决。我们已经确定了线粒体 基质蛋白C1qBP是一种新的CypD结合蛋白，我们的初步数据表明它可以 抑制MPT和细胞死亡。相反，我们发现促死亡激酶GSK3可以使细胞对 MPT和死亡，并且这些效应与CypD的磷酸化有关。因此，我们的中央 假设C1qBP抑制，而GSK3促进MPT和细胞死亡通过直接 CypD的调控。本申请的目的是利用遗传、生化、生理 以及系统评价CypD抑制剂(C1qBP)和CypD作用的药理学技术 激活剂(GSK3)在MPT、心肌细胞死亡和心肌疾病进展中的作用。在目标1中，我们将 确定C1qBP在MPT反应和心肌细胞死亡中的功能参与。在《目标2》中，我们 将评估依赖GSK3的CypD磷酸化对MPT和心肌细胞死亡的影响。这个 这项拟议研究的基本原理是，一旦调节MPT和MPT的关键线粒体蛋白 线粒体的功能被确定，它们可以作为一种靶向治疗一系列人类 心脏病。

PROJECT SUMMARY Mitochondrial dysfunction is an underlying cause of cardiomyocyte death and therefore plays a critical role in the development of many cardiac pathologies. This mitochondrial dysfunction is often mediated by the opening of the mitochondrial permeability transition (MPT) pore, which causes a rapid increase in inner mitochondrial membrane permeability. This in turn leads to ATP depletion, reactive oxygen species production, mitochondrial swelling and rupture. Consequently, our long-range goal is to identify the proteins that make up the MPT pore and understand the molecular mechanisms by which this complex is regulated. The MPT pore was originally proposed to consist of the voltage-dependent anion channel (VDAC) in the outer membrane, the adenine nucleotide translocase (ANT) in the inner membrane, plus a regulatory protein cyclophilin-D (CypD) in the matrix. However, genetic studies have revealed that VDAC and ANT are dispensable for MPT. Thus CypD still remains as the only bona fide member of the MPT pore complex. Importantly, the key factors that directly regulate CypD and its pro-MPT function remain to be elucidated. In particular, CypD-binding proteins that act to inhibit CypD's function have yet to be identified and the additional role that CypD phosphorylation plays in regulating MPT and cell death has not been comprehensively addressed. We have identified the mitochondrial matrix protein C1qbp as a novel CypD-binding protein and our strong preliminary data indicate that it can inhibit MPT and cell death. Conversely, we have found that the pro-death kinase GSK3 can sensitize cells to MPT and death, and that these effects are associated with CypD phosphorylation. Consequently, our central hypothesis is that C1qbp inhibits, whereas GSK3 promotes, MPT and cell death through the direct regulation of CypD. The objective of the present application is to utilize genetic, biochemical, physiological, and pharmacological techniques to systematically evaluate the roles of a CypD inhibitor (C1qbp) and a CypD activator (GSK3) in MPT, cardiac cell death, and the progression of myocardial disease. In Aim 1 we will determine the functional involvement of C1qbp in the MPT response and cardiac myocyte death. In Aim 2 we will evaluate the effects of GSK3-dependent CypD phosphorylation on MPT and cardiac cell death. The rationale for the proposed research is that once key mitochondrial proteins that regulate MPT and mitochondrial function are identified, they can be targeted as a means of treating a whole array of human cardiac diseases.