Regulation, signaling, and dynamics of glucan phosphatases.

葡聚糖磷酸酶的调节、信号传导和动力学。

• 批准号: 8642327
• 负责人: Matthew S. Gentry
• 金额: $ 0.81万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8642327
• 关键词: 5' -AMP-activated protein kinase; Affect; Binding; Biological Assay; CNS degeneration; Carbohydrates; Cardiac; Cells; Cessation of life; Complex; Comprehension; Crystallography; Deterioration; Deuterium; Disease; Energy Metabolism; Enzymes; Epilepsy; Event; Family suidae; Genes; Glucans; Glycogen; Glycogen (Starch) Synthase; Glycogen Debranching Enzyme; Grant; Human; Human body; Hydrogen; In Vitro; Knowledge; Lafora Disease; Life; Link; Mammalian Cell; Mass Spectrum Analysis; Metabolism; Modification; Molecular; Monitor; Motor; Mutation; Myocardium; Myoclonic Epilepsies; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Parkinson Disease; Patients; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Progressive Myoclonic Epilepsies; Proteins; Regulation; Review Literature; Role; Seizures; Series; Signal Transduction; Single Seizures; Skeletal Muscle; Specificity; Structure; Symptoms; Therapeutic; Tonic - clonic seizures; Ubiquitin; Ubiquitination; Water; Wolff-Parkinson-White Syndrome; Wolves; Work; base; design; glycogen metabolism; in vivo; insight; loss of function; loss of function mutation; member; mouse model; novel; overexpression; protein protein interaction; public health relevance; research study; sensor; tissue/cell culture; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The objective of this grant is to elucidate the regulation, downstream signaling, and structural dynamics of the glucan phosphatase laforin. Mammalian cells store readily mobilized energy in the form of glycogen, a water-soluble carbohydrate. Recessive mutations in genes encoding the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin disrupt glycogen metabolism and result in a fatal, neurodegenerative epilepsy called Lafora disease (LD). A hallmark of LD is poorly branched, hyperphosphorylated insoluble carbohydrate accumulations called Lafora bodies (LBs), thought to be the causative agent of LD. We established that malin is a single-subunit E3 ubiquitin ligase that ubiquitinates and triggers the degradation of multiple proteins involved in glycogen metabolism. In addition, we discovered that laforin is the founding member of a unique class of phosphatases that dephosphorylate phospho-glucans. These results allowed us to propose molecular mechanisms that cause LD: 1) loss of malin results in LBs due to an imbalance in glycogen metabolism proteins, i.e. protein levels are not properly maintained; 2) loss of laforin results in LBs due to glucan hyperphosphorylation that inhibits glucan branching; 3) laforin also acts as a targeting subunit for malin, so that loss of laforin disrupts some malin-directed ubiquitination events. While we have made significant strides in determining the molecular mechanisms of LD, we lack an understanding of how these enzymes are regulated. This proposal will elucidate the deficiencies in our current knowledge. We recently identified novel phosphorylation and ubiquitination events on laforin. Phosphorylation and ubiquitination are post-translational modifications that direct changes in protein concentration, enzymatic activity, protein localization, protein-protein interactions, and structural dynamics. In Aim 1 we will define the in vivo conditions triggering laforin phosphorylation and characterize the functional consequences. We will utilize 1) overexpression and endogenous protein levels in tissue culture cells to monitor the status of laforin localization and modification, 2) assay laforin function in vitro using purified proteins, and 3) utilize a LD mouse model to verify our results. In Aim 2, we will utilize a similar strategy to determine the affects of ubiquitination on laforin function. In addition, we will define the role of laforin in malin directed ubiquitination, as we have recently discovered that laforin acts as a targeting protein for malin. In Aim 3, we will determine how perturbations of the structural components of glucan phosphatases contribute to LD. We will utilize Hydrogen- Deuterium exchange mass spectrometry to define the structural dynamics of laforin and x-ray crystallography to determine the structure of a glucan phosphatase. This proposal is built on our past discoveries and uses complementary approaches to advance our understanding of the intercalated events of cell metabolism, neurodegeneration, and epilepsy. Completion of this work will yield a better understanding of these complex events and will produce therapeutic insights for epilepsy and neurodegeneration.

描述（由申请人提供）：该授权的目的是阐明葡聚糖磷酸酶laforin的调控、下游信号传导和结构动力学。哺乳动物细胞以糖原（一种水溶性碳水化合物）的形式储存容易调动的能量。编码E3泛素连接酶malin或双特异性磷酸酶laforin的基因的隐性突变破坏糖原代谢，并导致称为Lafora病（LD）的致命性神经退行性癫痫。LD的一个标志是分支不良、过度磷酸化的不溶性碳水化合物积聚，称为Lafora小体（LB），被认为是LD的病原体。 我们确定malin是一种单亚基E3泛素连接酶，它泛素化并引发参与糖原代谢的多种蛋白质的降解。此外，我们发现laforin是一类独特的磷酸酶的创始成员，这些磷酸酶使磷酸葡聚糖脱磷酸化。这些结果使我们能够提出导致LD的分子机制：1）由于糖原代谢蛋白质的不平衡，即蛋白质水平没有得到适当的维持，malin的损失导致LB; 2）由于抑制葡聚糖分支的葡聚糖过度磷酸化，laforin的损失导致LB; 3）laforin还充当malin的靶向亚基，使得laforin的缺失破坏了一些malin定向的泛素化事件。虽然我们在确定LD的分子机制方面取得了重大进展，但我们缺乏对这些酶如何调节的理解。这个建议将阐明我们目前知识的不足。 我们最近发现了laforin上新的磷酸化和遍在化事件。磷酸化和泛素化是翻译后修饰，其指导蛋白质浓度、酶活性、蛋白质定位、蛋白质-蛋白质相互作用和结构动力学的变化。在目标1中，我们将定义触发laforin磷酸化的体内条件并表征功能后果。我们将利用1）组织培养细胞中的过表达和内源性蛋白水平来监测laforin定位和修饰的状态，2）使用纯化的蛋白在体外测定laforin功能，以及3）利用LD小鼠模型来验证我们的结果。在目标2中，我们将利用类似的策略来确定泛素化对laforin功能的影响。此外，我们将定义laforin在malin定向泛素化中的作用，因为我们最近发现laforin作为malin的靶向蛋白。在目标3中，我们将确定葡聚糖磷酸酶的结构组分的扰动如何导致LD。我们将利用氢-氘交换质谱法来确定拉福林的结构动力学，并利用X射线晶体学来确定葡聚糖磷酸酶的结构。这项建议是建立在我们过去的发现，并使用互补的方法来推进我们的细胞代谢，神经退行性变和癫痫的插入事件的理解。这项工作的完成将产生对这些复杂事件的更好理解，并将产生对癫痫和神经变性的治疗见解。