Impact of naturally occurring osmolytes on protein structure and energetics

描述(申请人提供)：对渗透调节物质的特性混合物在正常身体功能中的作用以及在许多疾病中的作用缺乏了解，包括糖尿病和癌症。长期目标是了解这些渗透分子在人类中的正常和异常作用，并设计在疾病治疗和预防中使用渗透分子的策略。该项目的目标是了解非特定(优先)相互作用如何有助于在体内观察到渗透分子混合物的明显必要性。假设蛋白质、核苷酸配体和渗透分子的水溶液混合物对复合渗透分子鸡尾酒的需求是固有的。其基本原理是，这项研究为理解渗透分子混合物在许多重要的正常和异常细胞过程中所起的作用提供了基础。这些包括肾和脑的正常组织功能，以及渗透激素在许多疾病中的病理影响。核心假说将通过追求具体目标来检验，这些目标解决了为什么复杂的渗透分子混合物可能是必要的主要原因：渗透分子对蛋白质和核苷酸的不同影响，这导致了混合物的必要性；以及渗透分子的非相加作用，这导致了复杂的非线性行为。渗透分子对蛋白质-核苷酸系统的影响将通过高通量方法进行量化。热熔化和有效浓度的测量将在微板上进行，并将使用常规方法来验证结果。最初的模型蛋白是腺苷酸激酶、二氢叶酸还原酶和碳酸氢酶，以及小核苷酸配体，包括ATP、AMP、NADP等。人类和整个自然界中常见的13个渗透分子将被用于二元混合物。这个项目的创新之处在于，它的想法是，病理性渗透分子混合物是许多致命疾病的一个促成因素。我们预计，了解天然渗透压鸡尾酒对蛋白质和核苷酸的影响将有助于区分有害和有益的混合物，并阐明渗透压在许多疾病中的作用。这些信息对于确定通过药物、补充剂或饮食来操纵细胞渗透物质含量的靶点将是非常有价值的。这个项目意义重大，因为它克服了理解细胞病理生理学的一个主要障碍，即当渗透分子在其天然混合物中与蛋白质和代谢物结合时，会出现哪些新的性质，从而打开了改善疾病的新机会。这类研究的其他好处包括增强对蛋白质折叠、蛋白质结晶和药物配方的理解。 与公共健康相关：这个项目调查渗透物质(自然存在于人类中的小分子)的混合物，以及其中一些鸡尾酒如何对生物分子产生有益或有害的影响。预计这些结果将有助于寻找有效的化学预防药物来预防各种癌症和其他渗透调节物质起作用的疾病。

DESCRIPTION (provided by applicant): There is a lack in understanding about the role of characteristic mixtures of osmolytes in normal body function, and in many disease conditions, including diabetes mellitus and cancer. The long-term goal is to understand the normal and abnormal roles of these osmolytes in humans, and to design strategies for the use of osmolytes in the treatment and prevention of disease. This project's objective is to understand how non-specific (preferential) interactions contribute to the apparent necessity of osmolyte mixtures observed in vivo. It is hypothesized that the requirement for complex osmolyte cocktails is intrinsic to aqueous mixtures of proteins, nucleotide ligands, and osmolytes. The rationale is that this research provides the basis for understanding the role that osmolyte mixtures play in so many important normal and abnormal cellular processes. These include normal tissue function in kidney and brain, and the pathological impact of osmolytes in many diseases. The central hypothesis will be tested by pursuing Specific Aims that address the major reasons why complex osmolyte mixtures may be necessary: disparate impact of osmolytes on proteins and nucleotides, which leads to the necessity of the mixtures; and non-additive action of osmolytes, which leads to complex, non-linear behavior. The impact of osmolytes on protein-nucleotide systems will be quantified through high-throughput methods. Thermal melts and measurements of effective concentrations will be performed on microplates, and conventional approaches will be used to verify the results. The initial model proteins are adenylate kinase, dihydrofolate reductase, and carbonic anhydrase, in conjunction with small nucleotide ligands, including ATP, AMP, NADP, and others. Thirteen osmolytes that are common in humans and throughout nature will be used in binary mixtures. This project is innovative in its idea that pathological osmolyte mixtures are a contributing factor to many deadly diseases. We expect that knowledge of the effects of natural osmolyte cocktails on proteins and nucleotides will allow differentiating between deleterious and beneficial mixtures, and elucidate osmolytes' contributing role in many diseases. Such information would be very valuable in identifying targets for the manipulation of the osmolyte content of cells through medication, supplements, or diet. This project is significant, because it overcomes a major block in understanding cellular pathophysiology, namely the question of what new properties emerge when osmolytes are combined in their natural mixtures with proteins and metabolites, thus opening new opportunities of ameliorating disease. Additional benefits of this kind of research include an enhanced understanding of protein folding, of protein crystallization, and of drug formulation. PUBLIC HEALTH RELEVANCE: This project investigates mixtures of osmolytes (small molecules that naturally occur in humans), and how some of these cocktails can lead to beneficial or detrimental effects on biomolecules. It is expected that these results will facilitate the search for efficient chemoprevention agents against various cancers, and other diseases in which osmolytes play a role.