ROLE OF OXIDIZED CARDIOLIPIN TRANSLOCATION IN OXIDATIVE STRESS-INDUCED APOPTOSIS

描述（由申请人提供）：在病理性氧化应激条件下，即当内源性抗氧化剂被氧化压力淹没时，哺乳动物细胞可以发生凋亡或程序性细胞死亡。例如，在心脏细胞中，氧化诱导的细胞凋亡与缺血/再灌注损伤和蒽环类药物引起的心肌病等疾病有关。凋亡信号通常始于线粒体，在那里进行氧化能量代谢。呼吸链细胞色素c （cyt c）从线粒体内膜（IM）释放并移动到细胞质中是已知的内在凋亡途径的早期事件。静息状态cyt - c通过与心肌磷脂（一种仅存在于此腔室的磷脂）的相互作用与IM结合。由于高度不饱和，CL可以被氧化修饰成氢过氧化物（clohs）， clohs缺乏结合cyt - c的能力，比未氧化的CL更亲水。基于这一发现以及我们在模型膜系统中有关cloh易位和促凋亡多肽tBid结合的初步发现，我们提出以下假设：IM中CL的过氧化促进了其自发或蛋白质介导的外膜易位，在那里它招募tBid和随后形成低聚物的Bax，以产生可通过cyt c的孔。为了验证这一假设，我们计划在(1)脂质体IM/OM模型系统中研究膜间cloh转移与cyt - c释放和tBid/Bax靶向/渗透的关系，(2)测量关联/解离动力学和结合常数；(3)氧化修饰的有丝分裂体和线粒体；(4)氧化应激心肌细胞。机械推理将通过使用线粒体定位抗氧化剂，即过表达MitoGPx4和给药MitoQ来辅助。尖端的分析技术，如biacore为基础的表面等离子体共振，高效液相色谱与电化学检测，高效薄层色谱与磷成像检测将在项目中使用。这些新颖和创新的研究将为氧化诱导细胞凋亡的内在（线粒体中心）途径的早期事件提供重要的新的机制见解。考虑到与氧化应激相关的许多病理状况，包括几种心血管疾病，其中许多都以细胞凋亡性组织损伤为特征，该研究在生物医学上也具有重要意义。

DESCRIPTION (provided by applicant): Mammalian cells can undergo apoptosis or programmed cell death under pathologic oxidative stress conditions, i.e. when endogenous antioxidants are overwhelmed by oxidative pressure. In heart cells, for example, oxidant-elicited apoptosis is observed in connection with disorders such as ischemia/reperfusion injury and anthracycline-induced cardiomyopathy. Apoptotic signaling often begins in mitochondria, where oxidative energy metabolism takes place. Release of respiratory chain cytochrome c (cyt c) from the mitochondrial inner membrane (IM) and movement into cytosol is known to be an early event in the intrinsic apoptotic pathway. Resting state cyt c is bound to IM via interaction with cardiolipin (CL), a phospholipid located exclusively in this compartment. Being highly unsaturated, CL can undergo oxidative modification to hydroperoxide species (CLOOHs), which lack the ability to bind cyt c and are more hydrophilic than non-oxidized CL. Based on this and our preliminary findings pertaining to CLOOH translocation and binding of the proapoptotic polypeptide tBid in a model membrane system, we present the following hypothesis: Peroxidation of CL in the IM facilitates its spontaneous or protein-mediated translocation to the outer membrane (OM), where it recruits tBid and thence oligomer-forming Bax for generation of cyt c-traversable pores. Our plan for testing this hypothesis is to study intermembrane CLOOH transfer in relation to cyt c release and tBid/Bax targeting/permeabilization in (1) liposomal IM/OM model systems with (2) measurements of association/dissociation kinetics and binding constants; (3) oxidatively modified mitoplasts and mitochondria; and (4) oxidatively stressed cardiomyocytes. Mechanistic deductions will be assisted by use of mitochondria-localizing antioxidants, viz. overexpressed MitoGPx4 and administered MitoQ. Cutting-edge analytical techniques such as Biacore-based surface plasmon resonance, high-performance liquid chromatography with electrochemical detection, and high- performance thin layer chromatography with phosphorimaging detection will be used in the project. These novel and innovative studies will provide important new mechanistic insights into early events in the intrinsic (mitochondrion-centered) pathway of oxidant-induced apoptosis. The research is also biomedically significant, considering the numerous pathological conditions associated with oxidative stress, many of which, including several cardiovascular disorders, are characterized by apoptogenic tissue damage.