Impact of ALS-linked mutations on the structure, dynamics and function of profilin-1

ALS 相关突变对 profilin-1 结构、动力学和功能的影响

基本信息

批准号：
10323045
负责人：
Daryl Angela Bosco
金额：
$ 52.11万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-08 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10323045
关键词：
3-Dimensional Actin-Binding Protein Actins Affect Affinity Amyotrophic Lateral Sclerosis Binding Binding Proteins Binding Sites Biochemical Biological Biological Assay Biophysics Categories Cells Cellular biology Chimera organism Communication Complex Computer Simulation Coupling Cytoskeletal Proteins Data Defect Diagnosis Disease Exhibits Fluorescence Spectroscopy Gene Mutation Genes In Vitro Inherited Link Mammalian Cell Measures Molecular Conformation Motion Motor Neurons Mutation Neurodegenerative Disorders Pathogenesis Pathway interactions Phenotype Play Population Process Proline Protein Dynamics Protein Family Protein Region Proteins Publishing RNA Processing Relaxation Research Research Personnel Resolution Roentgen Rays Role Solubility Specificity Stress Fibers Structure Symptoms Testing Thermodynamics Variant Work base experimental study falls in silico innovation link protein member molecular dynamics multidisciplinary mutant novel polymerization profilin profilin 1 protein aggregation proteostasis screening small molecule trafficking

项目摘要

MASSI/ BOSCO – ABSTRACT Profilin-1 (PFN1) is a 15kDa actin-binding protein that plays a critical role in regulating cytoskeletal dynamics. In order to promote actin polymerization in cells, PFN1-bound actin must bind and synergize with formin proteins. In 2012, we identified mutations in PFN1 that cause the uniformly lethal neurodegenerative disease amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease. Defects in cytoskeletal dynamics and trafficking were already implicated in ALS pathogenesis, however the mechanism(s) by which ALS-linked proteins disrupt these processes is poorly understood. Many in the field predicted that ALS-linked mutations in PFN1 would abrogate the binding of PFN1 to other cytoskeletal proteins, such as actin and/or members of the formin family of proteins. However, rather unexpectedly, our extensive preliminary data show the opposite. ALS-linked PFN1 (ALS-PFN1) exhibits enhanced binding affinity for select formin proteins in cells and in vitro. Further, ALS-PFN1 causes formins to become hyperactivated, leading to accelerated and greater actin polymerization in cells. Our previous biochemical analyses demonstrated that ALS-PFN1 variants are severely destabilized and prone to aggregate. However, the overall three-dimensional x-ray structures of PFN1 mutants are very similar to WT PFN1. Therefore, we hypothesize that altered protein dynamics caused by ALS- mutations account for our observed phenotypes with respect to formin binding, actin polymerization and PFN1 aggregation. To test this hypothesis, Drs. Francesca Massi and Daryl Bosco have formed a multi- disciplinary, collaborative project that combines molecular dynamics (MD), NMR, fluorescence spectroscopy and cell biology to study the interactions of PFN1 with both formins and actin at atomistic resolution. Indeed, our novel preliminary MD simulations indicate that ALS-linked mutations perturb a network of residues within PFN1 that contact both actin and formin. Further, our extensive preliminary NMR data reveal PFN1 residues near the formin-binding interface are affected by actin binding, and that ALS-linked mutations perturb the intrinsic dynamics of PFN1. Collectively, our preliminary data provide a strong premise that altered protein dynamics of ALS-PFN1 contributes to dysregulated actin polymerization and protein aggregation, which are both involved in ALS pathogenesis. Our proposal builds upon our exciting preliminary data to rigorously characterize the dynamics and mechanism(s) of binding between PFN1, actin and formin. These approaches are highly innovative in the context of studying actin dynamics, and are ideal for probing the actin-PFN1-formin ternary complex, for which little is known at atomistic resolution. These studies are expected to have a significant and broad impact on neurodegenerative disease research, as well as on our fundamental understanding of actin dynamics.

Massi/ Bosco - 摘要 Profilin-1（PFN1）是一种15KDA肌动蛋白结合蛋白，在调节细胞骨架动力学中起关键作用。为了促进细胞中的肌动蛋白聚合，PFN1结合的肌动蛋白必须结合并与formin协同作用蛋白质。 2012年，我们确定了PFN1中引起均匀致命神经退行性疾病的突变肌萎缩性侧索硬化症（ALS），也称为Lou Gehrig病。细胞骨架动力学和贩运已经与ALS发病机理有关，但是ALS连接的机制蛋白质破坏了这些过程的理解很少。该领域的许多人预测ALS连接突变 PFN1将废除PFN1与其他细胞骨架蛋白的结合，例如肌动蛋白和/或成员蛋白质家族。但是，相当出乎意料的是，我们广泛的初步数据恰恰相反。 ALS连接的PFN1（ALS-PFN1）表现出对细胞和体外选择formin蛋白的结合亲和力增强。此外，ALS-PFN1导致formins变得过度活化，导致加速和更大的肌动蛋白细胞中的聚合。我们以前的生化分析表明，ALS-PFN1变体非常严重不稳定并容易汇总。但是，PFN1突变体的总体三维X射线结构与WT PFN1非常相似。因此，我们假设ALS-引起的蛋白质动力学改变了突变解释了我们观察到的表型相对于肌蛋白结合，肌动蛋白聚合和PFN1聚集。为了检验这一假设，博士。 Francesca Massi和Daryl Bosco已经形成了多种多样的结合分子动力学（MD），NMR，荧光光谱的纪律协作项目和细胞生物学，以原子分辨率研究PFN1与formins和肌动蛋白的相互作用。的确，我们的新型初步MD模拟表明，ALS连接的突变扰动了一个残留网络 PFN1接触肌动蛋白和follin。此外，我们广泛的初步NMR数据揭示了PFN1残差近形结合界面附近受肌动蛋白结合的影响，并且与ALS连接的突变扰动 PFN1的固有动力学。总的来说，我们的初步数据提供了改变蛋白质的强烈前提 ALS-PFN1的动力学有助于肌动蛋白聚合和蛋白质聚集的失调，这是两者都参与ALS发病机理。我们的建议基于我们令人兴奋的初步数据的严格性表征PFN1，肌动蛋白和follin之间结合的动力学和机制。这些方法在研究肌动蛋白动力学的背景下是高度创新的，是探测肌动蛋白PFN1形式的理想选择三元络合物，在原子分辨率上鲜为人知。这些研究预计将有一个对神经退行性疾病研究的重大影响，以及我们的基本了解肌动蛋白动力学。