HAX-1: a Multifaceted Family of Apoptotic Regulators

描述（由申请人提供）：HAX-1在约10年前被鉴定为HS 1的结合伴侣，HS 1是一种参与T细胞成熟的蛋白质。在其氨基端存在两个Bcl同源（BH）结构域，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，我们和其他人已经证明，HAX-1通过抑制起始caspase-9和死亡caspase-3的活化，并通过其与肌内质网Ca 2 + ATP酶（SERCA）泵及其调节剂受磷蛋白（PLN）的直接相互作用，促进Ca 2+稳态的调节，从而促进细胞存活。重要的是，这些先前的研究仅集中在变体1上，即在几个物种中大量表达的原型HAX-1蛋白。然而，最近的证据表明，HAX-1基因是大量剪接，产生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，我们的实验室着手检查大鼠心肌中HAX-1变体的存在和性质。使用RT-PCR分析和2D凝胶电泳，我们确认了至少七种HAX-1亚型（变体I-变体VII）的存在。在大鼠心脏H9 C2细胞培养物中瞬时转染变体（v）vI-vVII并结合亚细胞分级分离，表明它们编码分子量约为35-20 kDa的蛋白质，靶向线粒体和SR膜。变体I，V，VI和VII在用不同刺激诱导细胞凋亡后表现出显著的抗细胞凋亡活性，而变体II，III和IV加剧了细胞死亡，其中VIV是最有效的。虽然VIV本身在H9 C2心肌细胞中的过表达并不影响它们的活力，但它显著增强了细胞对凋亡损伤的反应。总之，我们的研究结果表明，HAX-1包括一个亚家族的蛋白质驻留在线粒体和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对凋亡刺激的存活。为了保持我们的研究重点，我们计划检查的属性的促凋亡VIV的抗凋亡VI，显示最有效的促/抗凋亡活动。因此，我们将调查的机制途径，通过HAX-1 vIV可能会反对vI的抗凋亡活性，使用分子，细胞和生物化学方法的组合（目的1），并检查是否在体内下调vIV通过腺相关病毒介导的RNA干扰，可以恢复心脏的形态和功能，在大鼠压力超负荷通过横向主动脉缩窄（目的2）。 拟议的研究将显着扩大我们目前的知识，在调节细胞存活和细胞死亡的HAX-1蛋白质亚家族的各种活动，以及它们作为心脏疾病的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，在晚期HF中，左心室辅助装置或心脏移植是最终选择。在过去的10年中，分子和细胞干预已经被探索，包括基因和细胞治疗方法。我们的研究将提供关于一种新的蛋白质亚家族，即HAX-1，在调节心肌细胞存活和死亡中的作用及其作为心脏病治疗靶点的潜在用途的重要信息。

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.