Mechanisms of amyloid nucleation

淀粉样蛋白成核机制

• 批准号: 8216635
• 负责人: RONALD B WETZEL
• 金额: $ 28.79万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8216635
• 关键词: Affect; Amides; Amyloid; Amyloid Fibrils; Amyloidosis; Applications Grants; Area; Bacterial Adhesins; Biological Assay; Biological Process; Biology; Biotechnology; Cell Nucleus; Cells; Cellular biology; Charge; Complex; Crowding; Cytoplasmic Granules; DNA; DNA Sequence Rearrangement; Data; Dependence; Disease; Drug Formulations; Elements; Enzymes; Equation; Equilibrium; Event; Exhibits; Growth; Hormones; Hydrogen Bonding; Inclusion Bodies; Indium; Insulin; Kinetics; Length; Literature; Mammals; Methods; Microbial Biofilms; Modeling; Molecular; Mutation; Nature; Neurodegenerative Disorders; Normal Cell; Organism; Pattern; Peptides; Pharmacologic Substance; Play; Polymer Chemistry; Polymers; Positioning Attribute; Preparation; Process; Property; Protein Biosynthesis; Proteins; Reaction; Recombinant Proteins; Recombinants; Role; Series; Sodium Chloride; Solutions; Sorting - Cell Movement; Staging; Stress; Structural Models; Structure; System; Temperature; Testing; Therapeutic Agents; Thermodynamics; Time; Urea; Vertebral column; Work; amyloid fibril formation; amyloid formation; amyloid structure; base; biological systems; design; disulfide bond; driving force; expectation; falls; human disease; monomer; normal aging; novel strategies; polyglutamine; polymerization; protein aggregate; protein aggregation; protein expression; protein folding; protein function; protein structure; research study; success

DESCRIPTION (provided by applicant): The aggregation of peptides and proteins into highly organized aggregates such as amyloid fibrils is a very important and fundamental process in biology. Most proteins suffer some degree of aggregation after they are synthesized in the cell, and a variety of cellular mechanisms have evolved to deal with the consequences of aggregation, which range from rendering protein synthesis inefficient to creating toxic species. In some cases amyloid fibril formation is beneficial and necessary for a particular biological function, such as the bacterial adhesins that support biofilm growth, and the secretory storage granules used by mammals for storage and release of protein hormones like insulin. In many other cases amyloid fibril formation is detrimental, being associated with serious human diseases as well as with normal aging. Protein aggregation also plays important roles in biotechnology, in the recombinant synthesis, purification and formulation of protein therapeutic agents. For all of these reasons it is important to come to a better mechanistic understanding of the protein aggregation process, and in particular the events that initiate aggregation, a process termed nucleation. Many cases of spontaneous amyloid fibril formation involve highly complex nucleation mechanisms with multiple molecular species in high flux, greatly complicating nucleation analysis. In other cases, however, nucleation is more simple, and in fact more resembles the classical model of nucleated growth polymerization from basic polymer chemistry. This grant application proposes a series of experiments to unravel the nucleation mechanism of a series of modified polyglutamine (polyQ) molecules, which are an important place to begin to study nucleation because they fall into the second, more simple class of nucleation mechanisms. The basic approach will be to design polyQ sequences containing various mutations that are predicted to have defined conformational effects on the solution structure of the protein, and to carry out detailed nucleation kinetics analyses on these proteins. These analyses will yield important parameters such as the thermodynamic driving force for nucleus formation, and the thermodynamics associated with simple fibril growth (elongation). These experimentally determined values for the energetics of amyloid nucleation and growth will then be compared with expectations based on the known conformational tendencies introduced into the test polyQ molecules by the added mutations. In this way, certain hypotheses for the structural nature of the aggregation nucleus will be tested. Success in this project will provide important clues as to how amyloid formation is nucleated that should have implications over broad areas of biology, such as biological systems affected by amyloid diseases, as well as related protein aggregation processes that occur in normal cell biology and in normal aging. Since it is likely that conformational rearrangements are important even in the more complex type of amyloid nucleation reactions, the results may open the way to new approaches to the study of the more complex and currently largely impenetrable class of nucleation mechanisms. PUBLIC HEALTH RELEVANCE: Proteins are the workhorses of the cell and are the agents by which the blueprints encoded in an organisms DNA are carried out. But proteins, as polymers, have a biophysical tendency to aggregate that is often in competition with the evolved function of the protein to fold into an active enzyme, hormone, etc. In some cases the aggregation ability of certain proteins has been utilized by nature in a positive way to enhance function. But aggregation is most of the time detrimental, being an aspect of normal aging, and a recurring feature of a number of important neurodegenerative diseases and other diseases. Aggregation also causes major problems in biotechnology. This project is designed to study in detail the mechanisms by which protein aggregation is initiated, including a possible intimate relationship to the mechanism of normal protein folding. The results will have wide impact for the understanding of normal and abnormal biology as well as the manufacturing and formulation of protein pharmaceuticals.

描述（由申请人提供）：肽和蛋白质聚集成高度组织化的聚集体，如淀粉样纤维，是生物学中非常重要和基本的过程。大多数蛋白质在细胞中合成后都会发生一定程度的聚集，并且已经进化出各种细胞机制来处理聚集的后果，这些机制包括使蛋白质合成效率低下到产生有毒物质。在某些情况下，淀粉样蛋白原纤维的形成对于特定的生物功能是有益的和必要的，例如支持生物膜生长的细菌粘附素，以及哺乳动物用于储存和释放蛋白质激素如胰岛素的分泌性储存颗粒。在许多其他情况下，淀粉样蛋白原纤维的形成是有害的，与严重的人类疾病以及正常衰老有关。蛋白质聚集在生物技术中，在蛋白质治疗剂的重组合成、纯化和配制中也起重要作用。由于所有这些原因，重要的是要更好地理解蛋白质聚集过程的机理，特别是引发聚集的事件，称为成核的过程。许多自发性淀粉样蛋白原纤维形成的情况涉及高度复杂的成核机制，具有高通量的多个分子种类，使成核分析大大复杂化。然而，在其他情况下，成核更简单，实际上更类似于基础聚合物化学中的成核生长聚合的经典模型。该授权申请提出了一系列实验来解开一系列修饰的聚谷氨酰胺（polyQ）分子的成核机制，这是开始研究成核的重要地方，因为它们属于第二类更简单的成核机制。基本的方法将是设计polyQ序列包含各种突变，预测有确定的构象影响的蛋白质的溶液结构，并进行详细的成核动力学分析这些蛋白质。这些分析将产生重要的参数，如核形成的热力学驱动力，以及与简单原纤维生长（伸长）相关的热力学。然后将这些实验确定的淀粉样蛋白成核和生长的能量学值与基于通过添加的突变引入测试polyQ分子中的已知构象趋势的预期值进行比较。以这种方式，某些假设的结构性质的聚集核将被测试。该项目的成功将为淀粉样蛋白的形成提供重要的线索，这将对广泛的生物学领域产生影响，例如受淀粉样蛋白疾病影响的生物系统，以及正常细胞生物学和正常衰老中发生的相关蛋白质聚集过程。由于它是可能的，构象重排是重要的，即使在更复杂的类型的淀粉样蛋白成核反应，结果可能开辟了新的方法来研究更复杂的，目前在很大程度上无法穿透类的成核机制。 公共卫生关系：蛋白质是细胞的主要工作者，也是生物体DNA中编码的蓝图得以实现的媒介。但是，蛋白质作为聚合物，具有聚集的生物物理倾向，这通常与蛋白质的进化功能竞争，以折叠成活性酶，激素等。在某些情况下，某些蛋白质的聚集能力已被自然界以积极的方式利用，以增强功能。但聚集在大多数时候是有害的，是正常衰老的一个方面，也是许多重要的神经退行性疾病和其他疾病的复发特征。聚集也造成生物技术方面的重大问题。该项目旨在详细研究蛋白质聚集启动的机制，包括与正常蛋白质折叠机制的可能密切关系。这些结果将对正常和异常生物学的理解以及蛋白质药物的制造和配制产生广泛的影响。