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: 8832738
• 负责人: Yuxin Mao
• 金额: $ 28.32万
• 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/8832738
• 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; DNA Sequence Alteration; Data; Diabetes Mellitus; Dimerization; Disease; Employee Strikes; Enzymes; Event; Family; 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; 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，例如细胞信号传导、增殖、细胞骨架的组织、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、腓骨肌萎缩症 (CMT) 和肌萎缩侧索硬化症 (ALS)。一类 PI 代谢酶包含一个名为 Sac 的保守 PI 磷酸酶模块。尽管受到相当多的关注，但人们对这个 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 结构域更喜欢特定的 PI 亚组作为底物，但如何通过其他同源的 Sac 结构域来确定底物特异性尚未解决。此外，酶活性的调节机制仍然很大程度上未知。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、催化功能和调节以及家族内多样性的分子机制。为了实现这一目标，我们最近解析了酵母 Sac1 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构型和催化位点处的大带正电凹槽。 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，并且Sac结构域的二聚化可能在功能调节中发挥作用。基于这些结构特征，我们建议进一步探究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Sac1的膜相互作用及界面催化机制； (3)阐明Sac1酶活性的调控机制。我们将采用多学科方法，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实现我们的具体目标。我们期望通过我们的长期努力，我们将获得关于这个包含 Sac 结构域的磷酸酶家族功能的分子基础的新知识。鉴于这些酶在正常细胞功能中发挥管家作用，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。与公共卫生的相关性还来自于以下事实：病原菌中的一些PI磷酸酶被发现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能力。 。建立囊磷酸酶的分子基础对于理解其基本细胞生物学非常有价值，并且还可以为内源性基因突变或外源性病原体感染条件下的治疗干预提供候选靶标。

项目成果

The structure of phosphoinositide phosphatases: Insights into substrate specificity and catalysis.

• DOI: 10.1016/j.bbalip.2014.09.015
• 发表时间: 2015-06
• 期刊: Biochimica et biophysica acta
• 作者: Hsu F;Mao Y
• 通讯作者: Mao Y

The Sac domain-containing phosphoinositide phosphatases: structure, function, and disease.

含 Sac 结构域的磷酸肌醇磷酸酶：结构、功能和疾病。

• DOI: 10.1007/s11515-013-1258-y
• 发表时间: 2013-08
• 期刊: Frontiers in biology

Crystal structure of Legionella pneumophila dephospho-CoA kinase reveals a non-canonical conformation of P-loop.

嗜肺军团菌去磷酸 CoA 激酶的晶体结构揭示了 P 环的非规范构象。

• DOI: 10.1016/j.jsb.2014.10.008
• 发表时间: 2014
• 期刊: Journal of structural biology
• 影响因子: 3
• 作者: Gong,Xiaojian;Chen,Xiaofang;Yu,Dongmin;Zhang,Nannan;Zhu,Zhongliang;Niu,Liwen;Mao,Yuxin;Ge,Honghua
• 通讯作者: Ge,Honghua