Solving the Phase Problem of TDP-43 and ALS-Associated Variants

解决 TDP-43 和 ALS 相关变体的相位问题

• 批准号: 10252768
• 负责人: Nikaela Whitney Bryan
• 金额: $ 3.33万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10252768
• 关键词: 3-Dimensional; Adhesives; Adopted; Affect; Amino Acids; Amyloid Fibrils; Amyotrophic Lateral Sclerosis; Biochemical Reaction; Biological; Biological Assay; Biology; Brain Stem; C-terminal; Cells; Chemicals; Code; Complex; Crystallization; Cues; Cytoplasmic Inclusion; DNA-Binding Proteins; Deuterium; Development; Disease; Environment; Foundations; Goals; Hydrogen; In Vitro; Individual; Ionic Strengths; JAM protein; Knowledge; Lead; Length; Link; Liquid substance; Mass Spectrum Analysis; Membrane; Messenger RNA; Methods; Molecular; Molecular Conformation; Motor Cortex; Motor Neuron Disease; Motor Neurons; Muscle Weakness; Mutation; N-terminal; Nature; Neurologic; Organelles; Pathologic; Patients; Phase; Play; Problem Solving; Property; Protein C; Protein Conformation; Protein Dynamics; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Recombinants; Reporting; Resolution; Respiratory Failure; Response Elements; Risk; Role; Spinal Cord; Structure; Testing; Therapeutic; Thermodynamics; Time; Transact; Variant; analytical method; aqueous; base; cost; delta protein; design; experimental study; flexibility; in vivo; inhibitor/antagonist; mutant; novel therapeutics; protein B; protein TDP-43; protein aggregation; protein folding; protein misfolding; protein structure; recruit; tool

One of biology's central conundrums is how the cell regulates complex biochemical reactions in time and space. Cells have solved this problem by producing compartments, namely organelles, comprised of distinct chemical environments. Emerging evidence suggests that multiple "membrane-less" organelles within the cell assemble via phase separation and behave like droplets within an aqueous environment. In vivo and in vitro, such phase separation is primarily driven by intrinsically disordered domains or proteins (IDPs). The structural plasticity of IDPs allows them to adopt various structural conformations, generating multiple, weakly adhesive, inter- and intramolecular interactions. Unfortunately, this conformation flexibility comes at the cost of an increased risk of protein jamming or aggregation. Evidence suggests that imbalances between the thermodynamic drive to undergo phase separation and the established opposing aggregation control machinery could lead to disease. For example, amyotrophic lateral sclerosis (ALS) is believed to arise from aberrant phase separation of transactive response (TAR) element DNA binding protein of 43 kDa (TDP-43). TDP-43 is an essential RNA binding protein that plays a fundamental role in mRNA splicing, transport, and stability. Despite having a vast knowledge of the individual domains of TDP-43, there has been relatively little study of the full-length protein that is found in biologically relevant phase separation. In particular, the structural and sequence-specific basis of TDP-43 phase separation is not well understood and remains elusive. Our lab has a very successful track-record with incorporating hydrogen/deuterium exchange mass spectrometry (HXMS) to solve emerging biological questions. The overall objective of this proposal is to utilize HXMS to understand the structural and molecular cues that trigger TDP-43 phase separation. I hypothesize that there are small, 3-5 amino acid long, regions within the N-terminal, first RNA-recognition motif, and C-terminal domains of TDP-43 that are required for phase separation. Additionally, this proposal will look at linking the structural dynamics of ALS-disease variants with their increased propensity to phase separate. I hypothesize that certain mutations perturb the structure of the C-terminal domain of TDP-43, producing larger or additional contact points for self-association, leading to an increased degree of phase separation. My proposed experiments will provide a basic understanding of how full-length and variant TDP-43 behaves in solution and lay the foundation for the structure- based development of efficient inhibitors.

生物学的核心难题之一是细胞如何在时间和空间上调节复杂的生化反应。 细胞通过产生由不同的化学物质组成的隔室，即细胞器，解决了这个问题。 环境.新出现的证据表明，细胞内的多个“无膜”细胞器组装在一起， 通过相分离并在水性环境中表现得像液滴。在体内和体外，这种阶段 分离主要由固有无序结构域或蛋白质（IDP）驱动。结构塑性 IDP使它们能够采用各种结构构象，产生多个弱粘合剂， 分子内相互作用不幸的是，这种构象灵活性是以增加的风险为代价的。 蛋白质堵塞或聚集。有证据表明，热力学驱动与 经历相分离和建立的相反的聚集控制机制可能导致疾病。 例如，肌萎缩性侧索硬化症（ALS）被认为是由肌萎缩性侧索硬化症的异常相分离引起的。 43 kDa的反式反应（TAR）元件DNA结合蛋白（TDP-43）。TDP-43是一种重要的RNA 在mRNA剪接、转运和稳定性中起重要作用的结合蛋白。尽管有一个巨大的 尽管对TDP-43的各个结构域了解不多，但对 在生物学相关的相分离中发现。特别是，结构和序列特异性基础 TDP-43相分离的机理还没有很好地理解，仍然难以捉摸。我们的实验室有一个非常成功的 采用氢/氘交换质谱（HXMS）解决新兴的 生物问题。本提案的总体目标是利用HXMS了解结构和 触发TDP-43相分离的分子线索。我假设有小的，3-5个氨基酸长， TDP-43的N端、第一RNA识别基序和C端结构域内的区域， 用于相分离。此外，这项建议将着眼于连接ALS疾病的结构动力学， 变体具有增加的相分离倾向。我假设某些突变扰乱了 TDP-43的C-末端结构域的结构，产生更大或额外的接触点用于自缔合， 导致相分离程度增加。我提出的实验将提供一个基本的 了解全长和变体TDP-43在溶液中的行为，并为结构奠定基础- 开发高效缓蚀剂。