Structural and Functional Studies of the Sac Family Phosphoinositide Phosphatases

描述（由申请人提供）：磷酸肌苷（pi）控制许多细胞过程，如细胞信号传导、增殖、细胞骨架组织、膜运输、离子通道活性、转录和mRNA运输。PI代谢失调与许多人类遗传性疾病有关，包括某些癌症、糖尿病、劳氏综合征、双相情感障碍、肌萎缩侧索硬化症（ALS）和肌萎缩侧索硬化症（CMT）。一类PI代谢酶含有保守的PI磷酸酶模块Sac。尽管受到了广泛的关注，但人们对PI磷酸酶家族的分子特性知之甚少。例如，不同蛋白质中的Sac结构域偏好pi的特定亚群作为底物，但底物特异性如何由其他同源的Sac结构域决定尚不清楚。此外，调节酶活性的机制在很大程度上仍然是未知的。我们的总体研究目标是阐明这一重要PI磷酸酶家族的底物特异性、催化功能和调控以及家族内多样性的分子机制。为了实现这一目标，我们最近解决了酵母Sac1的保守Sac结构域的晶体结构，这是含有Sac结构域的磷酸酶家族的第一个结构。我们的Sac磷酸酶结构域的晶体结构揭示了催化基序的惊人配置和催化位点上的大正电荷槽。Sac结构域的晶体结构以及我们的初步生化数据也表明Sac磷酸酶可能形成二聚体，Sac结构域的二聚化可能在功能调控中发挥作用。基于这些结构特征，我们建议进一步研究Sac蛋白家族的分子机制，主要有以下三个具体目标：(1)阐明Sac磷酸酶的催化机制和底物特异性；(2)探究Sac1的膜间相互作用及界面催化机理；(3)阐明Sac1酶活性的调控机制。我们将运用多学科的方法，包括结构生物学、分子生物学、生物化学和细胞生物学工具来解决我们的具体目标。我们期待通过长期的努力，对Sac结构域磷酸酶家族功能的分子基础有新的认识。鉴于这些酶在正常细胞功能中起着看家的作用，并且这些酶的突变与神经元退行性疾病有关，我们也预计它们与人类健康密切相关。与公共卫生的相关性还来自以下事实：已发现致病菌中的一些PI磷酸酶被用作入侵宿主细胞并在宿主细胞中繁殖的“武器”，以及体内PI水平影响宿主细胞对病毒感染的防御。建立Sac磷酸酶的分子基础将对了解其基本细胞生物学非常有价值，也可能为内源性基因突变或外源性致病性感染条件下的治疗干预提供候选靶点。

DESCRIPTION (provided by applicant): Phosphoinositides (PIs) control numerous cellular processes such as cell signaling, proliferation, organization of cytoskeleton, membrane trafficking, ion channel activity, transcription and mRNA trafficking. Mis-regulated PI metabolism has been linked to a number of human hereditary diseases, including certain cancers, diabetes, Lowe's syndrome, Bipolar disorder, Charcot-Marie-Tooth disease (CMT) and Amyotrophic Lateral Sclerosis (ALS). One class of PI metabolizing enzymes contains a conserved PI phosphatase module named Sac. Despite considerable attention, little is known about the molecular properties of this family of PI phosphatases. For example, Sac domains in different proteins prefer a specific subgroup of PIs as substrates, but how the substrate specificity is determined by the otherwise homologous Sac domains is unresolved. Moreover, the mechanisms for the regulation of enzymatic activity are still largely unknown. Our overall Research Goal is to elucidate the molecular mechanisms underlying substrate specificity, catalytic function and regulation, and intra-family diversity of this essential PI phosphatases family. Towards this goal, we have recently solved the crystal structure of the conserved Sac domain from yeast Sac1, the first structure of the Sac domain-containing phosphatase family. Our crystal structure of the Sac phosphatase domain reveals a striking configuration of the catalytic motif and a large positively charged groove at the catalytic site. The crystal structure of the Sac domain, as well as our preliminary biochemical data also suggests that Sac phosphatases may form a dimer and the dimerization of Sac domain may play a role in functional regulation. Based on these structural features, we propose to further pursue the molecular mechanisms of the Sac protein family with the following three Specific Aims: (1) Delineate the catalytic mechanism and the substrate specificity of Sac phosphatases; (2) Probe the membrane interaction and the mechanism for interfacial catalysis of Sac1; (3) Elucidate the mechanism for the regulation of enzymatic activity of Sac1. We will apply a multi-disciplinary approach, including structural biology, molecular biology, biochemistry, and cell biology tools to address our specific aims. We expect with our long term efforts, we will gain new knowledge about the molecular basis for the function of this Sac domain-containing phosphatase family. Given that these enzymes play a house-keeping role in normal cellular function and that mutations of these enzymes are associated with neuronal degeneration diseases, we also anticipate a close Relevance to Human Health. The relevance to public health also comes from the fact that some PI phosphatases in pathogenic bacteria have been found to be used as "weapons" to invade and thrive in host cells and the fact that the in vivo PI levels affect host cell defense against viral infections. Establishing the molecular basis for the Sac phosphatases will be extremely valuable in the understanding of their basic cell biology and may also provide candidate targets for therapeutic intervention under conditions of endogenous genetic mutations or exogenous pathogenic infections.