Ionic Charge and pH Regulation of Apoptotic Signaling

本提案是对PAR-98-087的回应，“K08接受者的小额资助计划”，并与NIH K08 DK02537相关联。内皮细胞凋亡在溶血性尿毒症综合征和其他急性肾功能衰竭的发病机制中起重要作用。本研究将通过研究离子电荷和碱性条件抑制细胞色素c介导的caspase-3激活的分子机制，促进我们对内皮细胞凋亡的生物化学理解。我们已经开发了一种细胞色素c依赖的胞质caspase-3激活的体外实验，允许在该过程中的离散步骤被识别和表征。在这个实验中，添加到细胞质溶胶中的细胞色素c启动凋亡小体（细胞色素c的低聚物、凋亡激活因子-1和dATP/ATP）的形成，导致caspase-9的激活，随后激活caspase-3。我们发现，在正常的细胞内浓度下，碱性pH和阳离子都能抑制caspase-3的激活。过量的细胞色素c抑制了高pH的抑制作用，但不影响阳离子的抑制作用，这表明pH和阳离子强度在caspase-3激活的两个不同步骤中起作用。这项提议将检验从这些观察中得出的两个总体假设。首先，碱性pH影响凋亡细胞的形成，而离子电荷阻止caspase-9的激活。其次，非阳离子渗透溶质的细胞积累是caspase-3活化所需的阳离子强度降低所必需的。具体目的是：1)建立caspase-9的体外活化实验，确定碱度和高阳离子强度对caspase-9活化的影响；2)在高离子电荷和碱性条件下，采用尺寸分离柱研究凋亡细胞的形成和成分；3)检测非阳离子溶质积累在细胞凋亡中的作用，通过检测潜在的渗透物，如氨基酸、单糖和双糖，在我们的体外实验中干扰caspase-3的激活，并使用液相色谱法比较非凋亡细胞和凋亡细胞的细胞质中渗透溶质含量的差异。此资助亦有助提高申请人的研究效率、财政独立及职业发展，推动其成为独立研究者。

This proposal is in response to PAR-98-087, "Small grant program for K08 recipients," and is linked to NIH K08 DK02537. Endothelial cell apoptosis has an essential role in the pathogenesis of hemolytic uremic syndrome and other causes of acute renal failure. The studies in this proposal will advance our understanding of the biochemistry of endothelial cell apoptosis by examining the molecular mechanism by which ionic charge and alkaline conditions inhibit cytochrome c- mediated activation of caspase-3. We have developed an in vitro assay of cytochrome c-dependent activation of cytosolic caspase-3 allowing discrete steps in the process to be identified and characterized. In this assay cytochrome c added to cytosol initiates the formation of apoptosomes (oligomers of cytochrome c, apoptosis activating factor-1, and dATP/ATP) resulting in activation of caspase-9, which subsequently activates caspase-3. We showed that both alkaline pH and cations, at normal intracellular concentrations, inhibited activation of caspase-3. Excess cytochrome c suppressed the inhibition by high pH, but did not affect the inhibition by cations, suggesting that pH and cationic strength act at two different steps in caspase-3 activation. This proposal will test two overall hypotheses derived from these observations. First, that alkaline pH affects the formation of apoptosomes, whereas ionic charge prevents caspase-9 activation. Second, that cellular accumulation of a non-cationic osmotic solute is required for the decrease in cationic strength necessary for caspase-3 activation. The Specific Aims are 1) to develop an in vitro assay for caspase-9 activation, and determine the effect of alkalinity and high cationic strength on activation; 2) to study the formation and constituents of apoptosomes, using a size separation column, under high ionic charge and alkaline conditions; and 3) to examine the role of non- cationic solute accumulation in apoptosis by testing if potential osmoles, such as amino acids and mono- and di-saccharides, interfere with caspase-3 activation in our in vitro assay, and by comparing cytosol from nonapoptotic and apoptotic cells using liquid chromatography for differences in osmotic solute content. This grant will also enhance the research productivity, fiscal independence and career development of the applicant furthering his advancement towards becoming an independent investigator.