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

基本信息

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
项目类别：
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

项目摘要

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中的CAG密码子扩展引起，外显子1跨越蛋白产物，称为Httex1，是神经元核内的主要成分。 Diamond 实验室展示了 Y-27632 的标志性内含物，Y-27632 是 rho 相关激酶 (ROCK) 的小分子抑制剂。在果蝇和小鼠模型中，减少细胞中的 Httex1 聚集并改善 Httex1 介导的毒性被确定为 ROCK 的直接靶标，磷酸-profilin 不会减少 Httex1 聚集，而未磷酸化的 profilin 通过直接相互作用调节 Httex1 聚集。我们设想了一种直接治疗方法，涉及以下内容：设计分子来模拟 profilin 的作用，这种方法需要全面了解 profilin 抑制 Httex1 聚集的机制，这是我们提案的重点是了解 profilin 如何调节外显子 1 的聚集。我们的方法将包括结合荧光光谱、电子顺磁共振的细胞内聚集测定和体外生物物理研究。 Profilin 调节 Httex1 聚集的相关实体是 Httex1 中聚谷氨酰胺的 C 末端富含 38 个残基的脯氨酸延伸段，该延伸段包含通过 17 个氨基酸连接的两个聚脯氨酸模块。 -残基柔性接头。Profilin 与 C38 中的聚脯氨酸模块结合。数据显示，在 profilin 存在的情况下，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 优先与较小的寡聚体结合，我们的初步数据还表明，与单独的 C38 相比，profilin 对 Httex1 构建体的表观亲和力更高。 M 相中的 Htt-NTF 是指寡聚体中 C38 模块的局部浓度增加。我们将根据初步数据来揭示这一点。 Profilin 结合影响 Httex1 疾病相关 N 末端片段相行为的机制。