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.

生物学的中心难题之一是细胞如何调节时间和空间中的复杂生化反应。 细胞通过产生隔室（即细胞器）解决了这个问题，该室由不同的化学 环境。新兴的证据表明，细胞组装中的多个“无膜”细胞器 通过相位分离，在水性环境中表现得像液滴一样。在体内和体外，这种阶段 分离主要由本质上无序的域或蛋白质（IDP）驱动。结构可塑性 IDP允许他们采用各种结构构象，产生多种，弱粘合剂，间和 分子内相互作用。不幸的是，这种构象的灵活性是以增加的风险 蛋白质干扰或聚集。有证据表明，热力学驱动器之间的不平衡 进行相分离，并且已建立的相对聚集控制机制可能导致疾病。 例如，肌萎缩性外侧硬化症（ALS）被认为是由于异常相分离而产生的 43 kDa（TDP-43）的交易反应（TAR）元素DNA结合蛋白。 TDP-43是必不可少的RNA 结合蛋白在mRNA剪接，运输和稳定性中起着基本作用。尽管有广阔的 了解TDP-43的各个领域，对全长蛋白的研究相对较少 在生物学相关的相分离中发现。特别是结构和序列特定的基础 TDP-43相分离尚未得到充分理解，并且仍然难以捉摸。我们的实验室非常成功 结合氢/氘交换质谱（HXM）的轨道记录以解决新兴 生物学问题。该提案的总体目的是利用HXM来了解结构和 触发TDP-43相分离的分子提示。我假设有小的3-5氨基酸长， 所需的N端，第一RNA识别基序和C末端域内的区域。 用于相分离。此外，该提案将着眼于链接ALS-疾病的结构动态 变体具有增加相位分离的倾向。我假设某些突变会扰动 TDP-43的C末端结构域的结构，产生更大或其他接触点以进行自我关联， 导致相位分离的程度增加。我提出的实验将提供基本 了解溶液中的全长和变体TDP-43的表现，并为结构奠定基础 - 基于有效抑制剂的发展。