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 大约 10 年前被鉴定为 HS1 的结合伴侣，HS1 是一种参与成熟的蛋白质 T细胞。其 NH2 末端存在两个 Bcl 同源 (BH) 结构域表明它可能发挥关键作用 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 通过抑制起始 caspase-9 和死亡 caspase-3 的激活来促进细胞存活，并通过 通过与肌内质的直接相互作用，有助于调节 Ca2+ 稳态 网状 Ca2+ ATP 酶 (SERCA) 泵及其调节剂受磷蛋白 (PLN)。重要的是，之前的这些 研究仅集中于变体 1，即原型 HAX-1 蛋白，在 几个物种。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的功能活性的亚型。 鉴于这些观察结果，我们的实验室着手检查 HAX-1 的存在和特性 大鼠心肌的变异。通过 RT-PCR 分析和 2D 凝胶电泳，我们证实了 至少七种 HAX-1 同工型（变体 I-变体 VII）。培养物中变体 (v) vI-vVII 的瞬时转染 大鼠心脏 H9C2 细胞与亚细胞分离相结合表明它们编码的蛋白质 分子量约为 35-20 kDa，靶向线粒体和 SR 膜。变型 I、V、VI VII 和 VII 在不同刺激诱导细胞凋亡后发挥显着的抗凋亡活性，而 变体 II、III 和 IV 加剧了细胞死亡，其中 vIV 最为有效。尽管 vIV 过度表达 H9C2 心肌细胞本身并不影响其活力，但它显着增强了响应 细胞凋亡的侮辱。总而言之，我们的研究结果表明 HAX-1 包含一个位于以下位置的蛋白质亚家族： 线粒体和 SR 膜可能促进细胞存活或细胞死亡。我们因此 假设 HAX-1 蛋白可能以拮抗作用调节心肌细胞存活，以响应 凋亡刺激。为了保持我们的研究重点，我们计划检查促凋亡 vIV 的特性 与抗凋亡 vI 相关，其显示出最有效的促/抗凋亡活性。因此，我们 将研究 HAX-1 vIV 可能对抗 vI 抗凋亡活性的机制途径， 结合使用分子、细胞和生化方法（目标 1），并检查体内是否存在 通过腺相关病毒介导的 RNA 干扰下调 vIV，可能会恢复心脏功能 通过横向主动脉缩窄承受压力超负荷的大鼠的形态和功能（目标 2）。 拟议的研究将极大地扩展我们目前对各种活动的了解 HAX-1 蛋白亚家族在调节细胞存活和细胞死亡中的作用及其作为新型、 心脏病的治疗目标。