Mechanisms of amyloid nucleation

淀粉样蛋白成核机制

基本信息

批准号：
8638028
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
项目类别：
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/8638028
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

项目摘要

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.

描述（由申请人提供）：将肽和蛋白质的聚集到高度有组织的聚集体中，例如淀粉样蛋白原纤维是生物学中非常重要且基本的过程。大多数蛋白质在细胞中合成后都会遭受一定程度的聚集，并且各种细胞机制已经进化以应对聚集的后果，从呈现蛋白质合成效率低下到产生有毒物种。在某些情况下，淀粉样蛋白原纤维形成是有益的，对于特定的生物学功能是有益的，也是必要的，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胰岛素等蛋白质激素的分泌储存颗粒。在许多其他情况下，淀粉样蛋白原纤维形成是有害的，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，在蛋白质治疗剂的重组合成，纯化和制定中也起着重要作用。由于所有这些原因，重要的是要更好地了解蛋白质聚集过程，尤其是启动聚集的事件，这是一种称为成核的过程。许多自发淀粉样蛋白原纤维形成的病例涉及高通量中多个分子物种的高度复杂成核机制，这极大地使成核分析复杂化。然而，在其他情况下，成核更简单，实际上更类似于基本聚合物化学的成核生长聚合的经典模型。该赠款的应用提出了一系列实验，以揭示一系列改良的聚谷氨酰胺（PolyQ）分子的成核机制，它们是开始研究成核的重要场所，因为它们属于第二类，更简单的成核机制。基本方法是设计包含各种突变的PolyQ序列，这些突变被预测对蛋白质的溶液结构定义了构象作用，并对这些蛋白质进行了详细的成核动力学分析。这些分析将产生重要参数，例如核形成的热力学驱动力，以及与简单原纤维生长（延长）相关的热力学。然后，这些实验确定的淀粉样蛋白成核和生长能量的值将与基于添加突变引入测试PolyQ分子中的已知构象趋势的期望进行比较。以这种方式，将测试有关聚集核的结构性的某些假设。该项目的成功将提供有关如何对淀粉样蛋白形成的成核形成的重要线索，这些形成应该对广泛的生物学领域具有影响，例如受淀粉样蛋白疾病影响的生物系统以及在正常细胞生物学和正常生物学中发生的相关蛋白质聚集过程老化。由于即使在更复杂的淀粉样蛋白成核反应类型中，构象重排也很重要，因此结果可能为研究更复杂且目前在很大程度上无法穿透的成核机制的新方法开辟了道路。