Novel Model Systems Link Floridean Starch Metabolism to Lafora Disease

新型模型系统将佛罗里达淀粉代谢与拉福拉病联系起来

• 批准号: 7871627
• 负责人: Matthew S. Gentry
• 金额: $ 7.43万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7871627
• 关键词: Animalia; Binding; Biochemical; Biochemistry; Bioinformatics; Biological Models; CNS degeneration; Carbohydrates; Cellular biology; Cessation of life; Complement; Complex; Deterioration; Disease; Eimeria tenella; Energy-Generating Resources; Eukaryota; Event; Genes; Genetic; Glycogen; Grant; Human; Invertebrates; Knock-out; Lafora Disease; Life; Link; Mammalian Cell; Mammals; Metabolism; Modeling; Motor; Myoclonic Epilepsies; Neurodegenerative Disorders; Neurologic; Organism; Orthologous Gene; Paramecium tetraurelia; Patients; Phenotype; Phosphoric Monoester Hydrolases; Process; Progressive Myoclonic Epilepsies; Protista; Reporting; Role; Seizures; Signal Transduction Pathway; Single Seizures; Starch; System; Testing; Tetrahymena thermophila; Tonic - clonic seizures; Tonic Seizures; Toxoplasma gondii; Transgenic Organisms; Vertebrates; carbohydrate metabolism; genome sequencing; glycogen metabolism; loss of function mutation; mRNA Expression; new therapeutic target; novel; particle; polyglucosan; protein expression; research study; ubiquitin-protein ligase

The focus of this grant is to determine how laforin, the only phosphatase in Kingdom Animalia with a carbohydrate binding domain, regulates changes in glycogen metabolism in vertebrates and invertebrates. Humans develop insoluble aberrant glycogen particles, called Lafora bodies, as a result of an autosomal recessive neurodegenerative disorder known as Lafora disease (LD). LD is the result of loss of function mutations to either of the genes that encode the dual specific phosphatase laforin or the E3 ubiquitin ligase malin. LD is one of five major progressive myoclonus epilepsies (PMEs) and presents as a single seizure in the second decade of the patient's life; this single event is followed by progressive central nervous system degeneration, intellectual decline, severe motor deterioration, and finally death within ten years. Laforin is reported as only being conserved among vertebrates; however, I recently identified laforin orthologs in five unicellular eukaryotes, including Cyanidioschyzon merolae and Toxoplasma gondii. Additionally, the biochemical composition of LBs closely resembles that of floridean starch; an insoluble carbohydrate molecule synthesized by the same unicellular eukaryotes that have laforin. Thus, there is a direct correlation between the presence of laforin and synthesis of floridean starch amongst invertebrates. I propose that laforin is involved in degrading insoluble carbohydrates, either to utilize it as an energy source for invertebrates or to protect cellular integrity in mammals, and that laforin's role in these two processes is conserved from invertebrates to humans. To determine laforin's function in glycogen metabolism I will: 1) Perform rigorous phylogenomic and functional analyses of laforin's invertebrate orthologs. I will identify every invertebrate laforin ortholog via bioinformatics and will determine the mRNA expression, protein expression and subcellular localization of laforin in C. merolae and T. gondii. 2) Define laforin's target in floridean starch metabolism in Toxoplasma gondii. I will generate multiple transgenic T. gondii organisms, identify any phenotypes that these organisms manifest and employ these organisms in biochemical experiments to identify laforin's substrate or a signal transduction pathway that it modulates. Additionally, I will utilize a T. gondii laforin knockout line to test if other laforin orthologs complement any observed phenotype. 3) Characterize the role of floridean starch in the human myoclonic epilepsy Lafora disease. I will extend my findings from the invertebrate systems to the vertebrate system and LD. I will test any laforin substrate identified in the previous sections to see if it is also a substrate of laforin in the mammalian system. Additionally, I will fully characterize the interaction I previously identified between malin and laforin to determine both the cause and effect of this interaction. The findings from all three of my specific aims can be applied to identify new therapeutic targets to treat LD and possibly other polyglucosan diseases.

这笔赠款的重点是确定Laforin如何是王国Animalia中唯一的磷酸酶 碳水化合物结合结构域，调节脊椎动物和无脊椎动物中糖原代谢的变化。 正染色体，人类会形成不溶性异常的糖原颗粒，称为Lafora身体 被称为Lafora病（LD）的隐性​​神经退行性疾病。 LD是功能丧失的结果 编码双重特异性磷酸酶Laforin或E3泛素连接酶的基因突变 马林。 LD是五个主要进行的肌阵挛性癫痫（PME）之一，并作为单一癫痫发作 患者生命的第二个十年；这个单一事件之后是渐进的中枢神经系统 堕落，智力下降，严重的运动恶化，最后在十年内死亡。拉福林是 据报道仅在脊椎动物中保守；但是，我最近在五个 单细胞真核生物，包括Cyanidioschyzon Merolae和Toxoplasma Gondii。另外， LB的生化组成与佛罗里达淀粉的生化成分非常相似；不溶性碳水化合物 由具有Laforin的相同单细胞真核生物合成的分子。因此，存在直接相关性 在无脊椎动物中的Laforin的存在与佛罗里达淀粉的合成之间。我建议 Laforin参与降解不溶性碳水化合物，要么将其用作能源 无脊椎动物或保护哺乳动物中的细胞完整性，而拉福林在这两个过程中的作用是 从无脊椎动物到人类保守。为了确定拉福林在糖原代谢中的功能：1） 对Laforin无脊椎动物直系同源物进行严格的系统基因组和功能分析。我会识别的 每个无脊椎动物Laforin直系同源物都通过生物信息学，并将确定mRNA表达，蛋白质 Laforin在Merolae和T. gondii中的表达和亚细胞定位。 2）定义拉福林的目标 弓形虫弓形虫中的佛罗里达淀粉代谢。我将产生多个转基因T. gondii生物， 确定这些生物体现并在生化中使用这些生物的任何表型 识别Laforin底物或调节的信号转导途径的实验。另外，我 是否会利用T. gondii laforin敲除线来测试其他Laforin orthologs是否补充了任何观察到的 表型。 3）表征佛罗里达淀粉在人类肌阵挛性癫痫Lafora病中的作用。我 我的发现将从无脊椎动物系统扩展到脊椎动物系统和LD。我将测试任何拉福林 底物在前面的部分中确定，以查看它是否也是哺乳动物系统中Laforin的底物。 此外，我将充分表征我先前在Malin和Laforin之间发现的相互作用 确定这种相互作用的原因和影响。我所有三个特定目标的发现都可以 应用于确定新的治疗靶标，以治疗LD和其他多葡萄糖疾病。