HAX-1: a Multifaceted Family of Apoptotic Regulators

HAX-1：多方面的凋亡调节因子家族

基本信息

批准号：
8030970
负责人：
Aikaterini Kontrogianni-Konstantopoulos
金额：
$ 22.5万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-15 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8030970
关键词：
Affect Apoptosis Apoptotic Binding Binding Sites Biochemical Biological Assay Ca(2+)-Transporting ATPase Cardiac Cardiac Myocytes Cardiac Myosins Cardiology Caspase Cell Death Cell Fractionation Cell Survival Cell Therapy Cells Cessation of life Co-Immunoprecipitations Collaborations Comparative Study Dependovirus Down-Regulation Echocardiography Ensure Family Gene Transfer Genes Heart Heart Diseases Heart Transplantation Heart failure Histologic Homeostasis Human Hypoxia In Vitro Induction of Apoptosis Injection of therapeutic agent Intervention Knowledge Laboratories Left Letters Light Literature Macaca Mediating Medicine Membrane Membrane Potentials Methodology Mitochondria Molecular Monitor Morphology Mus Myocardium Myosin Light Chains Pan Genus Pathway interactions Peptides Pharmaceutical Preparations Play Process Property Propidium Diiodide Protein Isoforms Proteins Pump RNA RNA Interference RNA Splicing Rattus Regulation Reticulum Reverse Transcriptase Polymerase Chain Reaction Role Sarcoplasmic Reticulum Series Serotyping Simulate Staining method Stains Stimulus Stress System T-Lymphocyte Tail Technology Testing Time Tissues Transcript Transcriptional Regulation Transfection Tropism Two-Dimensional Gel Electrophoresis Variant Veins Viral adenoviral-mediated annexin A5 caspase-3 caspase-9 constriction gel electrophoresis gene therapy hemodynamics in vivo mitochondrial membrane molecular mass mortality mutant new therapeutic target novel overexpression phospholamban pressure promoter research study response therapeutic target ventricular assist device young adult

项目摘要

DESCRIPTION (provided by applicant): HAX-1 was identified ~10 years ago as a binding partner of HS1, a protein involved in the maturation of T-cells. The presence of two Bcl Homology (BH) domains in its NH2-terminus suggested that it might play key roles in mediating cell survival. Using in vitro and in vivo systems, we and others have demonstrated that HAX-1 promotes cell survival by inhibiting the activation of initiator caspase-9 and death caspase-3, and by contributing to the regulation of Ca2+ homeostasis, through its direct interaction with the SarcoEndoplasmic Reticulum Ca2+ ATPase (SERCA) pump and its regulator phospholamban (PLN). Importantly, these previous studies have solely focused on variant 1, the prototypical HAX-1 protein that is abundantly expressed in several species. Recent evidence, however, has indicated that the HAX-1 gene is heavily spliced, giving rise to a number of isoforms with distinct molecular compositions and possibly functional activities. In view of these observations, our laboratory set forth to examine the presence and properties of HAX-1 variants in rat myocardium. Using RT-PCR analysis and 2D gel electrophoresis, we confirmed the presence of at least seven HAX-1 isoforms (variant I-variant VII). Transient transfections of variants (v) vI-vVII in cultures of rat cardiac H9C2 cells combined with subcellular fractionation indicated that they encode proteins with molecular masses of ~35-20 kDa, that target to both the mitochondrial and SR membranes. Variants I, V, VI and VII exerted significant anti-apoptotic activity following induction of apoptosis with different stimuli, whereas variants II, III and IV exacerbated cell death, with vIV being the most potent. Although overexpression of vIV per se in H9C2 cardiocytes did not affect their viability, it markedly enhanced cell death in response to an apoptotic insult. Taken together, our findings indicate that HAX-1 comprises a subfamily of proteins residing in the mitochondrial and SR membranes that may promote either cell survival or cell death. We therefore hypothesize that HAX-1 proteins may act antagonistically to regulate cardiomyocyte survival in response to an apoptotic stimulus. To keep our studies focused, we plan to examine the properties of the pro-apoptotic vIV in relation to the anti-apoptotic vI, which display the most potent pro-/anti-apoptotic activities. Consequently, we will investigate the mechanistic pathways through which HAX-1 vIV may oppose the anti-apoptotic activity of vI, using a combination of molecular, cellular and biochemical methodologies (Aim 1), and examine if in vivo down-regulation of vIV through adeno associated viral mediated RNA interference, may restore cardiac morphology and function in rats subjected to pressure overload through transverse aortic constriction (Aim 2). The proposed studies will significantly extent our current knowledge on the diverse activities of the HAX-1 subfamily of proteins in regulating cell survival and cell death, and their potential use as novel, therapeutic targets for cardiac disease. PUBLIC HEALTH RELEVANCE: Heart failure (HF) remains a leading cause of mortality in the developing world. Current drug treatment has limited efficiency, and in advanced HF, left ventricular assist devices or heart transplantation are the ultimate options. In the last 10 years, molecular and cellular interventions have been explored including gene and cell therapy approaches. Our studies will provide important information about the roles of a novel subfamily of proteins, namely HAX-1, in regulating cardiomyocyte survival and death, and their potential use as therapeutic targets for cardiac disease.

描述（由申请人提供）：HAX-1在10年前被确定为HS1的结合伴侣，HS1是T细胞成熟的蛋白质。其NH2末端中存在两个BCL同源性（BH）结构域的存在表明，它可能在介导细胞存活中起关键作用。使用体外和体内系统，我们和其他人表明，HAX-1通过抑制引发剂Caspase-9和死亡caspase-3的激活，以及通过与Sarcoendoplasmic retpase（sercane）（Sercane pumpane（Serca）（Serca）（serca）pumpan（plockator）（ploscanter）（phoscanter andtrancan）的调节器（plockator）的调节器（plockator）的plockator（plockator）的调节器（通过其直接相互作用），通过抑制了Ca2+稳态的调节来促进细胞的存活。重要的是，这些先前的研究仅集中在变体1上，这是一种典型的HAX-1蛋白，在几种物种中大量表达。然而，最近的证据表明，HAX-1基因被大量剪接，从而产生许多具有不同分子组成和可能功能活性的同工型。鉴于这些观察结果，我们的实验室着手研究大鼠心肌中HAX-1变体的存在和特性。使用RT-PCR分析和2D凝胶电泳，我们确认存在至少七个HAX-1同工型（变体I变量VII）。在大鼠心脏H9C2细胞培养物中变体（V）VI-VVII的瞬时转染与亚细胞分级结合表明它们用〜35-20 kDa的分子质量编码蛋白质，它们靶向〜35-20 kDa，它们靶向线粒体和SR膜。变体I，V，VI和VII在诱导不同刺激的凋亡后发挥了明显的抗凋亡活性，而变体II，III和IV变体加剧了细胞死亡，VIV是最有效的。尽管H9C2心脏细胞中VIV本身的过表达不会影响其生存能力，但它显着增强了细胞死亡，以响应凋亡的侮辱。综上所述，我们的发现表明，HAX-1包括位于线粒体和SR膜中的蛋白质的亚科，这些蛋白质可能促进细胞存活或细胞死亡。因此，我们假设HAX-1蛋白可以拮抗作用以调节凋亡刺激响应于心肌细胞的存活。为了保持研究的重点，我们计划检查促凋亡的体内与抗凋亡VI有关的特性，该抗凋亡VI表现出最有效的促/抗凋亡活性。因此，我们将研究使用分子，细胞和生化方法的组合（目标1）的组合HAX-1 VIV可能会反对VI的抗凋亡活性的机理途径，并检查体内通过adeno相关的病毒介导的RNA介导的RNA干扰的体内调节，是否会恢复跨性心脏的缩减（通过递交型自变量）。拟议的研究将显着使我们目前对蛋白质中HAX-1亚科在调节细胞存活和细胞死亡方面的不同活性的了解，以及它们作为心脏病的新型治疗靶标的潜在用途。公共卫生相关性：心力衰竭（HF）仍然是发展中国家死亡率的主要原因。当前药物治疗的效率有限，在晚期HF，左心室辅助设备或心脏移植是最终的选择。在过去的10年中，已经探讨了分子和细胞干预措施，包括基因和细胞疗法方法。我们的研究将提供有关蛋白质新型亚科（即HAX-1）在调节心肌细胞存活和死亡中的作用的重要信息，以及它们作为心脏病的治疗靶标的潜在用途。