HAX-1: a Multifaceted Family of Apoptotic Regulators

HAX-1在大约10年前被确定为HS1的结合伙伴，HS1是一种参与细胞成熟的蛋白质 T细胞。在其NH2末端存在两个BH同源结构域，这表明它可能起关键作用 在调节细胞存活中的作用。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caspase-9和死亡caspase-3的激活来促进细胞存活，并通过 通过与肌浆网的直接相互作用，促进钙离子稳态的调节 网状Ca~(2+)-ATP酶(SERCA)泵及其调节蛋白(PLN)。重要的是，这些以前的 研究仅集中在变体1上，这是典型的HAX-1蛋白，在 几个物种。然而，最近的证据表明，HAX-1基因高度剪接，导致 到许多具有不同分子组成和可能的功能活性的异构体。 鉴于这些观察，我们的实验室着手检查HAX-1的存在和性质 大鼠心肌中的变异体。通过RT-PCR分析和2D凝胶电泳，我们证实了 至少7种HAX-1亚型(变体I-变体VII)。突变体(V)VI-VVII在培养上的瞬时转化 对大鼠心脏H9C2细胞的亚细胞分离表明，它们编码的蛋白与 分子质量约35-20 kDa，靶向线粒体膜和SR膜。变体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加剧了细胞死亡，VIV是最有效的。虽然Viv基因过表达 H9C2心肌细胞本身并不影响其活性，它显著增加了细胞的死亡 细胞凋零的侮辱。综上所述，我们的发现表明，HAX-1由驻留在 可能促进细胞存活或细胞死亡的线粒体和SR膜。因此，我们 假设HAX-1蛋白可能拮抗地调节心肌细胞的存活 细胞凋亡刺激。为了保持我们的研究重点，我们计划检查促凋亡VIV在 与抗凋亡VI有关，后者显示出最强的促/抗凋亡活性。因此，我们 将研究HAX-1VIV对抗VI抗凋亡活性的机制途径， 使用分子、细胞和生化方法的组合(目标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，可能会恢复心脏 通过腹主动脉横断缩窄压力超负荷的大鼠的形态和功能(目标2)。 拟议的研究将大大扩展我们目前对联合国儿童基金会不同活动的了解 HAX-1亚家族蛋白在调节细胞存活和细胞死亡中的作用及其作为新的潜在用途， 心脏病的治疗靶点。

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.