HAX-1: a Multifaceted Family of Apoptotic Regulators

描述(申请人提供)：HAX-1在大约10年前被鉴定为HS1的结合伙伴，HS1是一种参与T细胞成熟的蛋白质。在其NH2末端存在两个BH同源结构域，提示它可能在调节细胞存活中发挥关键作用。利用体外和体内系统，我们和其他人已经证明，HAX-1通过抑制启动caspase-9和死亡caspase-3的激活，并通过与肌浆网钙ATPase(SERCA)泵及其调节蛋白(PLN)的直接相互作用来促进钙稳态，从而促进细胞存活。重要的是，这些以前的研究只关注变体1，这是在几个物种中大量表达的典型HAX-1蛋白。然而，最近的证据表明，HAX-1基因被大量剪接，产生了许多具有不同分子组成和可能功能活性的亚型。鉴于这些观察，我们的实验室着手研究HAX-1变异体在大鼠心肌中的存在和性质。通过RT-PCR分析和双向凝胶电泳法，我们证实至少存在7种HAX-1亚型(变异体I-变异体VII)。(V)VI-VVII突变体在大鼠心脏H9C2细胞培养中的瞬时转染和亚细胞分离表明，它们编码的蛋白质分子质量约为35-20 kDa，以线粒体膜和SR膜为靶标。变异体I、V、VI和VII在不同刺激下诱导细胞凋亡后具有显著的抗凋亡活性，而变异体II、III和IV会加剧细胞死亡，其中VIV的作用最强。虽然在H9C2心肌细胞中过度表达病毒本身并不影响其活性，但它显著增加了细胞对凋亡损伤的反应。综上所述，我们的发现表明，HAX-1包括一个亚家族的蛋白质，存在于线粒体和SR膜上，可能促进细胞存活或细胞死亡。因此，我们假设HAX-1蛋白可能在心肌细胞对凋亡刺激的反应中发挥拮抗作用，调节心肌细胞存活。为了保持我们的研究重点，我们计划检查促凋亡的VIV与抗凋亡的VI的关系，后者显示出最强的促/抗凋亡活性。因此，我们将采用分子、细胞和生化方法相结合的方法(目标1)，研究HAX-1VIV可能对抗VI的抗凋亡活性的机制途径(目标1)，并检查在体是否通过腺相关病毒介导的RNA干扰下调VIV的表达，是否可以恢复通过腹主动脉横断收缩而承受压力超负荷的大鼠的心脏形态和功能(目标2)。这些拟议的研究将极大地扩展我们目前对HAX-1亚家族蛋白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新的治疗靶点的潜在用途。 公共卫生相关性：心力衰竭(HF)仍然是发展中国家死亡的主要原因。目前的药物治疗效果有限，对于晚期心力衰竭，左心室辅助装置或心脏移植是最终选择。在过去的10年里，人们探索了分子和细胞干预措施，包括基因和细胞治疗方法。我们的研究将提供一个新的蛋白质亚家族，即HAX-1，在调节心肌细胞生存和死亡中的作用，以及它们作为心脏病治疗靶点的潜在用途。

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.