HAX-1: a Multifaceted Family of Apoptotic Regulators

HAX-1：多方面的凋亡调节因子家族

• 批准号: 8206608
• 负责人: Aikaterini Kontrogianni-Konstantopoulos
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8206608
• 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

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.

HAX-1在大约10年前被鉴定为HS 1的结合伴侣，HS 1是一种参与细胞成熟的蛋白质， T细胞在其氨基端存在两个Bcl同源（BH）结构域，表明它可能在细胞凋亡中起关键作用。 介导细胞存活的作用。使用体外和体内系统，我们和其他人已经证明， HAX-1通过抑制启动子caspase-9和死亡caspase-3的激活以及通过 通过与肌内质网的直接相互作用，促进Ca 2+稳态的调节 网囊Ca 2 + ATP酶（SERCA）泵及其调节剂受磷蛋白（PLN）。重要的是，这些前 研究仅集中在变体1，即在大肠杆菌中大量表达的原型HAX-1蛋白。 几个物种。然而，最近的证据表明，HAX-1基因是大量剪接的，从而引起了 许多具有不同分子组成和可能的功能活性的同种型。 鉴于这些观察结果，我们的实验室着手研究HAX-1的存在和性质 大鼠心肌中的变异。利用RT-PCR分析和2D凝胶电泳，我们证实了 至少七种HAX-1同种型（变体I-变体VII）。变体（v）vI-vVII在培养物中的瞬时转染 大鼠心脏H9 C2细胞结合亚细胞分级显示，他们编码的蛋白质， 分子量约为35-20 kDa，靶向线粒体和SR膜。备选案文一、五、六 和VII在用不同刺激诱导凋亡后表现出显著的抗凋亡活性，而 变体II、III和IV加剧了细胞死亡，其中vIV是最有效的。尽管vIV的过度表达 H9 C2心肌细胞本身并不影响它们的活力，它显着增强细胞死亡， 凋亡损伤综上所述，我们的发现表明HAX-1包含一个蛋白质亚家族，该亚家族位于 线粒体和SR膜可以促进细胞存活或细胞死亡。因此我们 假设HAX-1蛋白可能拮抗性地调节心肌细胞存活， 凋亡刺激为了使我们的研究集中，我们计划在细胞凋亡中检测促凋亡vIV的特性。 与抗凋亡vI相关，其显示最有效的促/抗凋亡活性。因此我们 将研究HAX-1 vIV可能对抗vI的抗凋亡活性的机制途径， 使用分子、细胞和生物化学方法的组合（目的1），并检查是否在体内 通过腺相关病毒介导的RNA干扰下调vIV， 通过横向主动脉缩窄（Aim 2）对压力超负荷大鼠的形态和功能进行了研究。 拟议的研究将大大扩展我们目前对各种活动的了解， 本发明涉及调节细胞存活和细胞死亡的HAX-1亚家族蛋白，以及它们作为新的、 心脏病的治疗靶点。