Novel Protein Phosphatases

新型蛋白磷酸酶

• 批准号: 7599219
• 负责人: JACK E DIXON
• 金额: $ 33.04万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-10-01 至 2010-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7599219
• 关键词: Active Sites; Affect; Animals; Apoptosis; Award; Bacteria; Bioenergetics; Bioinformatics; Caenorhabditis elegans; Cell Culture Techniques; Cell physiology; Cells; Crystallization; Data; Deterioration; Differentiation and Growth; Disease; Drosophila genus; Energy Metabolism; Etiology; Eukaryota; Eukaryotic Cell; Event; Genome; Glucose; Grant; HTATIP gene; Human; Kinetics; Knock-out; Knockout Mice; Knowledge; Laboratories; Lafora Disease; Link; Mediator of activation protein; Metabolism; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Molecular; Molecular Profiling; Mus; Mutate; Mutation; Myoclonic Epilepsies; Names; Neurologic; Organelles; PTEN gene; Patients; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Progressive Myoclonic Epilepsies; Protein Tyrosine Phosphatase; Protein phosphatase; Publications; RNA Interference; Role; Running; Signal Pathway; Signal Transduction; Site; Starch; Structure; Techniques; Technology; Tissues; United States National Institutes of Health; cell motility; design; falls; glycogen metabolism; insulin secretion; member; novel; polyglucosan; tissue/cell culture; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Members of the protein tyrosine phosphatase (PTP) superfamily, all contain the highly conserved active site motif, Cys-x5- Arg (Cx5R), and are key mediators of a variety of cellular processes including growth, differentiation, motility, metabolism and programmed cell death. This proposal focuses on two novel PTPs whose functions are potentially linked to energy metabolism in the cell. The first two specific aims are directed at understanding laforin, a phosphatase mutated in the most severe form of progressive myoclonus epilepsy, Lafora's disease (LD). The hallmark of LD is the accumulation of starch-like polyglucosans called Lafora bodies. My laboratory has recently shown that malin, an E3 ligase also mutated in a subset of LD patients, interacts with and ubiquitinates laforin. Specific Aim 1 outlines our intentions to fully characterize laforin's phosphatase activity. We have optimized bacterial expression and purification of laforin for use in these kinetic analyses as well as for crystallization efforts to determine the first structure of laforin. Specific Aim 2 focuses on the modification of laforin by malin and the cellular consequences of this modification. Successful completion of these specific aims will give us a better understanding of the etiology of LD. The remaining three specific aims are designed to characterize FLIP, the first known mitochondrial PTP. Given the absence of precedent for PTPs in this organelle, and a nearly complete lack of known mitochondrial signaling pathways involving reversible phosphorylation, we will first perform a thorough characterization of PLIP in Specific Aim 3. This aim includes pinpointing PLIP to a specific submitochondrial compartment, establishing its mechanism of import, and determining a tissue expression profile. Specific Aim 4 is an effort to elucidate the mitochondrial function(s) of PLIP using RNAi strategies in tissue culture cells and conditional knock-out strategies in mice. PLIP's potential function in cell culture will be evaluated with respect to glucose stimulated insulin secretion (GSIS), apoptosis, and the general - bioenergetic status of mitochondria. Our Cre-lox knockout mouse strategy will afford us the opportunity of ablating PLIP expression in a variety of different tissues enabling us to evaluate our cell culture data in the context of the whole animal. Specific Aim 5 is designed to identify the cellular substrate(s) for PLIP, in part through leveraging our powerful RNAi technique in Drosophila S2 cells. Successful completion of Specific Aims 3-5 will give us a better understanding of PLIP's role in mitochondrial function and help to reveal mitochondria as important centers for cell signaling.

描述（由申请人提供）：蛋白质酪氨酸磷酸酶（PTP）超家族的成员都包含高度保守的活性位点基序，Cys-X5-ARG（CX5R），并且是各种细胞过程的关键介体，包括生长，差异，差异，运动，机动性，代谢性和程序性细胞死亡。该提案的重点是两个新型PTP，其功能可能与细胞中的能量代谢有联系。前两个具体目标是针对理解拉福林的，拉福林是一种以最严重的进行性肌阵癫痫，拉福拉氏病（LD）突变的磷酸酶。 LD的标志是称为Lafora身体的淀粉状聚葡萄糖的积累。我的实验室最近表明，在LD患者中也突变的E3连接酶Malin与Laforin相互作用并泛素。具体目标1概述了我们完全表征Laforin磷酸酶活性的意图。我们已经优化了Laforin的细菌表达和纯化，以用于这些动力学分析以及确定Laforin的第一个结构的结晶努力。特定的目标2着重于Malin对Laforin的修饰以及这种修饰的细胞后果。这些特定目标的成功完成将使我们更好地了解LD的病因。其余的三个特定目标旨在表征Flip，这是第一个已知的线粒体PTP。考虑到该细胞器中PTP的先例，几乎完全缺乏已知的线粒体信号传导途径，涉及可逆磷酸化，我们将首先在特定的目标3中对PLIP进行彻底的特征。该目标包括将PLIP精确定位到PLIP上，以对特定的Combisschondrial cart术，并确定其进口的特定机制，并确定其组织的表达机制。具体目标4是一种努力，使用RNAI策略在组织培养细胞中使用RNAi策略阐明PLIP的线粒体功能和小鼠的条件敲除策略。 PLIP在细胞培养中的潜在功能将根据葡萄糖刺激的胰岛素分泌（GSIS），凋亡和线粒体的一般生物能状态进行评估。我们的Cre-Lox敲除小鼠策略将使我们有机会在各种不同的组织中消融PLIP表达，从而使我们能够在整个动物的背景下评估我们的细胞培养数据。特定目标5旨在通过利用我们在果蝇S2细胞中强大的RNAi技术来确定PLIP的细胞底物。成功完成特定目标3-5将使我们更好地了解PLIP在线粒体功能中的作用，并有助于揭示线粒体作为细胞信号的重要中心。