ROLE OF OXIDIZED CARDIOLIPIN TRANSLOCATION IN OXIDATIVE STRESS-INDUCED APOPTOSIS

描述(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压垮时，哺乳动物细胞可以经历凋亡或程序性细胞死亡。例如，在心脏细胞中，观察到氧化剂诱导的细胞凋亡与缺血/再灌注损伤和蒽环类药物诱导的心肌病等疾病有关。凋亡信号通常始于线粒体，在那里发生氧化能量代谢。呼吸链细胞色素c(Cytc)从线粒体内膜(IM)释放并进入胞浆是内源性细胞凋亡途径的早期事件。静息状态细胞色素c通过与心磷脂(CL)的相互作用与IM结合，心磷脂(CL)是一种专门位于该隔室的磷脂。CL是高度不饱和的，可以进行氧化修饰，生成的过氧化氢物种(CLOOHs)缺乏与Cytc结合的能力，比未氧化的CL更亲水。基于这一点以及我们关于模型膜系统中促凋亡多肽TBID的ClOOH移位和结合的初步发现，我们提出了以下假设：IM中CL的过氧化促进其自发或蛋白质介导的移位到外膜(OM)，在那里它招募TBID并由此形成寡聚体Bax以产生细胞可穿透的孔。我们计划通过(1)脂质体IM/OM模型体系(2)结合/解离动力学和结合常数的测量；(3)氧化修饰的有丝分裂细胞和线粒体；(4)氧化应激的心肌细胞，研究膜间ClOOH转移与细胞色素c释放和TBID/Bax靶向/通透性的关系。机械性推断将通过使用线粒体定位抗氧化剂来辅助，即。过表达MitoGPx4和给予MitoQ。该项目将使用尖端分析技术，如基于Biacore的表面等离子体共振、带有电化学检测的高效液相色谱和带有磷成像检测的高效薄层色谱。这些新颖和创新的研究将为氧化剂诱导的细胞凋亡的内在(以线粒体为中心)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情况，包括几种心血管疾病，这项研究也具有生物医学意义，其中许多疾病的特点是细胞凋亡性组织损伤。

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.