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: 7684042
• 负责人: Matthew S. Gentry
• 金额: $ 19.24万
• 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/7684042
• 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（动物王国中唯一的磷酸酶）如何发挥作用 碳水化合物结合域，调节脊椎动物和无脊椎动物糖原代谢的变化。 由于常染色体的作用，人类产生了不溶性异常糖原颗粒，称为拉福拉小体 隐性神经退行性疾病，称为拉福拉病 (LD)。 LD是功能丧失的结果 编码双特异性磷酸酶 Laforin 或 E3 泛素连接酶的基因发生突变 马林。 LD 是五种主要的进行性肌阵挛癫痫 (PME) 之一，在 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 退化、智力衰退、运动能力严重衰退，最后在十年内死亡。拉福林是 据报道仅在脊椎动物中保守；然而，我最近在五种药物中发现了 laforin 直向同源物 单细胞真核生物，包括 Cyanidioschyzon merolae 和弓形虫。此外， LB 的生化成分与佛罗里达淀粉非常相似；不溶性碳水化合物 由与 Laforin 相同的单细胞真核生物合成的分子。因此，有直接的相关性 拉福林的存在与无脊椎动物中佛罗里达淀粉的合成之间的关系。我建议 Laforin 参与降解不溶性碳水化合物，或者将其用作能量来源 无脊椎动物或保护哺乳动物的细胞完整性，laforin 在这两个过程中的作用是 从无脊椎动物到人类都是保守的。为了确定 Laforin 在糖原代谢中的功能，我将：1) 对 Laforin 的无脊椎动物直系同源物进行严格的系统发育和功能分析。我会识别 通过生物信息学分析每个无脊椎动物 Laforin 直系同源物，并将确定 mRNA 表达、蛋白质 Laforin 在 C. merolae 和 T. gondii 中的表达和亚细胞定位。 2) 定义laforin的目标 弓形虫中的佛罗里达淀粉代谢。我将产生多种转基因弓形虫生物体， 识别这些生物体表现出的任何表型，并将这些生物体用于生化 鉴定 laforin 底物或其调节的信号转导途径的实验。另外，我 将利用弓形虫 laforin 敲除系来测试其他 laforin 直向同源物是否补充任何观察到的 表型。 3) 描述佛罗里达淀粉在人类肌阵挛性癫痫拉福拉病中的作用。我 将把我的发现从无脊椎动物系统扩展到脊椎动物系统和LD。我会测试任何laforin 前面部分中鉴定的底物，看看它是否也是哺乳动物系统中 laforin 的底物。 此外，我将充分描述我之前确定的 malin 和 laforin 之间的相互作用，以 确定这种相互作用的原因和结果。我所有三个具体目标的发现可以 可用于确定治疗 LD 和其他可能的聚葡萄糖疾病的新治疗靶点。