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压倒时，哺乳动物细胞可发生凋亡或程序性细胞死亡。例如，在心脏细胞中，观察到氧化剂引起的细胞凋亡与诸如缺血/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，在线粒体中发生氧化能量代谢。呼吸链细胞色素c（cyt c）从线粒体内膜（IM）释放并进入胞质溶胶是已知的内在凋亡途径中的早期事件。静息状态cyt c通过与心磷脂（CL）相互作用与IM结合，心磷脂（CL）是一种仅位于该隔室中的磷脂。由于高度不饱和，CL可以进行氧化修饰为氢过氧化物物质（CLOOH），其缺乏结合cyt c的能力并且比未氧化的CL更具亲水性。基于这一点和我们的初步研究结果有关的CLOOH易位和结合的促凋亡多肽TBID在模型膜系统中，我们提出了以下假设：CL在IM的过氧化促进其自发或蛋白质介导的易位到外膜（OM），在那里它招募TBID，从而寡聚体形成Bax细胞色素C-可穿越的孔的产生。我们检验这一假设的计划是研究与cyt c释放和tBid/Bax靶向/透化相关的膜间CLOOH转移，其中（1）脂质体IM/OM模型系统，（2）测量缔合/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;和（4）氧化应激的心肌细胞。机械扣除将通过使用线粒体定位抗氧化剂，即过表达的MitoGPx 4和MitoQ来辅助。该项目将采用Biacore表面等离子体共振、高效液相色谱电化学检测、高效薄层色谱荧光成像检测等尖端分析技术。这些新的和创新的研究将提供重要的新的机制的见解，在氧化剂诱导的细胞凋亡的内在途径（以蛋白质为中心）的早期事件。该研究也具有生物医学意义，考虑到与氧化应激相关的许多病理条件，其中许多，包括几种心血管疾病，其特征在于致炎性组织损伤。

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.