Structural and Functional Studies of the Sac Family Phosphoinositide Phosphatases

囊家族磷酸肌醇磷酸酶的结构和功能研究

• 批准号: 8259725
• 负责人: Yuxin Mao
• 金额: $ 28.49万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259725
• 关键词: Actins; Active Sites; Address; Affect; Amyotrophic Lateral Sclerosis; Attention; Binding; Biochemical; Biochemistry; Biological Assay; Bipolar Disorder; Catalysis; Catalytic Domain; Cell membrane; Cell physiology; Cells; Cellular biology; Charcot-Marie-Tooth Disease; Charge; Collaborations; Complex; Cytoskeleton; Data; Diabetes Mellitus; Dimerization; Disease; Employee Strikes; Enzymes; Event; Family; Gene Mutation; Genetic; Genetic Transcription; Goals; Health; Hereditary Disease; Homologous Gene; Housing; Human; In Vitro; Infection; Institutes; Integral Membrane Protein; Invaded; Ion Channel; Knowledge; Link; Lipids; Liposomes; Malignant Neoplasms; Mammals; Membrane; Membrane Protein Traffic; Messenger RNA; Metabolism; Molecular; Molecular Biology; Molecular Conformation; Mutation; Names; Nerve Degeneration; Nuclear; Oculocerebrorenal Syndrome; Organelles; Patients; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Plants; Play; Property; Protein Family; Protein Tyrosine Phosphatase; Proteins; Public Health; Regulation; Research; Role; Signal Transduction; Site-Directed Mutagenesis; Structure; Study models; Subgroup; Substrate Specificity; Testing; Therapeutic Intervention; Universities; Virus Diseases; Work; Yeasts; analog; base; design; dimer; flexibility; in vivo; inhibitor/antagonist; insight; interfacial; member; mutant; novel; pathogenic bacteria; prevent; public health relevance; structural biology; synaptojanin; tool; trafficking; weapons; yeast protein

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.

描述（由申请人提供）：磷酸肌醇（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、腓骨肌萎缩性侧索硬化症（CMT）和肌萎缩性侧索硬化症（ALS）。一类PI代谢酶含有保守的PI磷酸酶模块，称为Sac。尽管相当多的关注，很少有人知道这个家庭的PI磷酸酶的分子特性。例如，不同蛋白质中的Sac结构域偏好PI的特定亚组作为底物，但底物特异性如何由另外同源的Sac结构域决定尚不清楚。此外，酶活性的调节机制在很大程度上仍然未知。我们的总体研究目标是阐明底物特异性，催化功能和调节，以及这个重要的PI磷酸酶家族的家族内多样性的分子机制。为了实现这一目标，我们最近已经解决了保守的Sac结构域的晶体结构从酵母Sac 1，第一个结构的Sac结构域含有磷酸酶家族。我们的Sac磷酸酶结构域的晶体结构揭示了催化基序和催化位点处的大的带正电荷的凹槽的惊人配置。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节中起作用。基于这些结构特征，我们提出进一步研究Sac蛋白家族的分子机制，具体目标如下：（1）阐明Sac磷酸酶的催化机制和底物特异性;（2）探讨Sac 1的膜相互作用和界面催化机制;（3）阐明Sac 1的酶活性调节机制。我们将采用多学科的方法，包括结构生物学，分子生物学，生物化学和细胞生物学工具，以解决我们的具体目标。我们期望通过我们的长期努力，我们将获得新的知识的分子基础上的功能，这个囊域含有磷酸酶家族。鉴于这些酶在正常细胞功能中起着管家作用，并且这些酶的突变与神经元变性疾病有关，我们还预计与人类健康密切相关。与公共健康的相关性还来自以下事实：已发现病原菌中的一些PI磷酸酶被用作入侵宿主细胞并在宿主细胞中茁壮成长的“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是非常有价值的，在了解其基本的细胞生物学，也可能提供候选目标的内源性基因突变或外源性病原体感染的条件下的治疗干预。