Mechanisms of amyloid nucleation

描述(申请人提供)：多肽和蛋白质聚集成高度组织的聚集体，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某种程度的聚集，并且已经进化出各种细胞机制来处理聚集的后果，从使蛋白质合成效率低下到产生有毒物种。在某些情况下，淀粉样原纤维的形成对于特定的生物功能是有益的，也是必要的，例如支持生物膜生长的细菌粘附素，以及哺乳动物用来储存和释放蛋白质激素(如胰岛素)的分泌储存颗粒。在许多其他情况下，淀粉样原纤维的形成是有害的，与严重的人类疾病和正常衰老有关。蛋白质聚集在生物技术中也发挥着重要作用，在蛋白质治疗药物的重组合成、纯化和配方中也发挥着重要作用。由于所有这些原因，重要的是要更好地从机理上理解蛋白质聚集过程，特别是启动聚集的事件，这一过程称为成核。许多自发形成淀粉样原纤维的案例涉及高度复杂的成核机制，在高通量下有多个分子物种，极大地增加了成核分析的复杂性。然而，在其他情况下，成核更简单，事实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。这项拨款申请提出了一系列实验来解开一系列改性聚谷氨酰胺(PolyQ)分子的成核机制，这些分子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包含各种突变的多聚Q序列，这些突变被预测为对蛋白质的溶液结构具有确定的构象影响，并对这些蛋白质进行详细的成核动力学分析。这些分析将产生重要的参数，如核形成的热力学驱动力，以及与简单纤维生长(伸长)相关的热力学。这些实验确定的淀粉样蛋白成核和生长的能量学数值随后将与基于已知构象趋势的预期值进行比较，这些趋势是通过添加的突变引入测试多Q分子的。这样，关于聚集核结构性质的某些假设将得到检验。该项目的成功将为淀粉样蛋白的形成提供重要的线索，这将对生物学的广泛领域产生影响，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。由于构象重排即使在更复杂类型的淀粉样核化反应中也很可能是重要的，这一结果可能会为研究更复杂且目前基本上难以理解的成核机制开辟新的途径。

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.