Mechanism of modulation of huntingtin exon 1 aggregation by profilin

Profilin 调节亨廷顿外显子 1 聚集的机制

• 批准号: 9107118
• 负责人: MARC I DIAMOND
• 金额: $ 60.55万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107118
• 关键词: Affinity; Avidity; Behavior; Binding; Biological Assay; C-terminal; CAG repeat; Caliber; Cells; Codon Nucleotides; Data; Diamond; Disease; Drosophila genus; Electron Microscopy; Electron Spin Resonance Spectroscopy; Equilibrium; Exons; Fluorescence Spectroscopy; Genes; Goals; Huntington Disease; Huntington gene; In Vitro; Lead; Length; Mediating; Messenger RNA; Mitosis; N-terminal; Neurodegenerative Disorders; Neurons; Nuclear Inclusion; Nuclear Protein; Phase; Phosphorylation; Population; Proline; Protein Fragment; Proteins; Proteolysis; RNA Splicing; Research Personnel; Rho-associated kinase; S Phase; Serine; Stretching; Therapeutic; Therapeutic Intervention; Thermodynamics; Toxic effect; Transcript; Translations; Work; beta pleated sheet; biophysical analysis; cytotoxicity; design; flexibility; insight; kinase inhibitor; light scattering; monomer; mouse model; mutant; polyglutamine; polyproline; prevent; profilin; public health relevance; small molecule inhibitor; targeted treatment

 DESCRIPTION (provided by applicant): Huntington's disease (HD) is a devastating neurodegenerative disease caused by CAG codon expansion in exon 1 of the huntingtin (htt) gene. Exon 1 spanning protein products, referred to as Httex1 are the major components of neuronal intranuclear inclusions that are the hallmarks of HD. Y-27632, a small molecule inhibitor of the rho-associated kinase (ROCK), was shown by the Diamond lab to reduce Httex1 aggregation in cells and ameliorate Httex1-mediated toxicity in Drosophila and mouse models. Serine-137 of profilin was established as the direct target of ROCK. Phospho-profilin does not reduce Httex1 aggregation whereas unphosphorylated profilin modulates Httex1 aggregation through direct interactions thus explaining the effect of Y-27632. We envisage a direct therapeutic approach that involves the design of molecules to mimic the effects of profilin. Such an approach requires a comprehensive understanding of the mechanisms by which profilin suppresses Httex1 aggregation, and this is the focus of our proposal. Our goal is to understand how profilin modulates the aggregation of exon 1 of huntingtin through interactions with its polyproline regions. Our approaches will include intracellular assays of aggregation and in vitro biophysical studies that combine fluorescence spectroscopies, electron paramagnetic resonance spectroscopy, and electron microscopy. The relevant entity for modulation of Httex1 aggregation by profilin is the 38-residue proline-rich stretch (C38) that is C-terminal to polyglutamine in Httex1. This stretch encompasses two polyproline modules that are connected via a 17-residue flexible linker. Profilin binds to the polyproline modules in C38. Our preliminary data show that in the presence of profilin, a higher total concentration of Htt-NTFs is required to form large spherical and fibrillar aggregates because profilin binds preferentially to smaller oligomeric species. Our preliminary data also establish that the apparent affinity of profilin for Httex1 constructs is higher when compared to C38 alone. This appears to be due to increased avidity that derives from oligomerization of Htt-NTFs in the M-phase. Avidity refers to the increased local concentration of C38 modules within oligomers. We will build on our preliminary data to uncover the mechanisms by which profilin binding impacts the phase behavior of disease- relevant N-terminal fragments of Httex1.

 描述（申请人提供）：亨廷顿病（HD）是一种破坏性神经退行性疾病，由亨廷顿（htt）基因外显子1中CAG密码子扩增引起。外显子1跨蛋白产物，称为Httex 1是神经元核内包涵体的主要成分，是HD的标志。Y-27632是rho相关激酶（ROCK）的小分子抑制剂，Diamond实验室显示它可以减少细胞中的Httex 1聚集，并改善果蝇和小鼠模型中Httex 1介导的毒性。确定profilin的丝氨酸-137为ROCK的直接靶点。磷酸化profilin不减少Httex 1聚集，而未磷酸化profilin通过直接相互作用调节Httex 1聚集，从而解释了Y-27632的作用。我们设想了一种直接的治疗方法，包括设计分子来模拟profilin的作用。这种方法需要全面了解profilin抑制Httex 1聚集的机制，这是我们建议的重点。我们的目标是了解profilin如何通过与其多聚脯氨酸区域的相互作用来调节亨廷顿蛋白外显子1的聚集。我们的方法将包括细胞内聚集测定和结合联合收割机荧光光谱、电子顺磁共振光谱和电子显微镜的体外生物物理研究。通过profilin调节Httex 1聚集的相关实体是Httex 1中多聚谷氨酰胺C-末端的38个残基的富含脯氨酸的片段（C38）。该延伸段包括通过17个残基的柔性接头连接的两个聚脯氨酸模块。Profilin与C38中的聚脯氨酸模块结合。我们的初步 数据显示，在存在profilin的情况下，需要更高的Htt-NTF总浓度， 形成大的球形和纤维状聚集体，因为profilin优先结合较小的寡聚物种类。我们的初步数据还确定，与单独的C38相比，Profilin对Httex 1构建体的表观亲和力更高。这似乎是由于M期中Htt-NTF的寡聚化引起的亲合力增加。亲合力是指寡聚物内C38模块的局部浓度增加。我们将建立在我们的初步数据，以揭示profilin结合影响疾病相关的N-末端片段的Httex 1的相行为的机制。