Impact of naturally occurring osmolytes on protein structure and energetics

描述（由申请人提供）：缺乏对渗透剂特征混合物在正常身体功能和许多疾病（包括糖尿病和癌症）中的作用的了解。长期目标是了解这些渗透剂在人类中的正常和异常作用，并设计使用渗透剂治疗和预防疾病的策略。本项目的目的是了解非特异性（优先）相互作用如何有助于在体内观察到的渗透剂混合物的明显必要性。据推测，对复杂的渗压剂混合物的需求是蛋白质、核苷酸配体和渗压剂的水性混合物所固有的。基本原理是，这项研究提供了理解的作用，渗透混合物在许多重要的正常和异常的细胞过程中发挥的基础。这些包括肾脏和大脑中的正常组织功能，以及渗透剂在许多疾病中的病理影响。将通过追求特定目标来检验中心假设，这些特定目标解决了可能需要复杂的渗透剂混合物的主要原因：渗透剂对蛋白质和核苷酸的不同影响，这导致了混合物的必要性;以及渗透剂的非累加作用，这导致了复杂的非线性行为。渗透剂对蛋白质-核苷酸系统的影响将通过高通量方法进行定量。将在微孔板上进行热熔和有效浓度测量，并使用常规方法验证结果。最初的模型蛋白是腺苷酸激酶、二氢叶酸还原酶和碳酸酐酶，与小的核苷酸配体（包括ATP、AMP、NADP等）结合。在人类和整个自然界中常见的13种渗透剂将以二元混合物的形式使用。这个项目的创新之处在于它认为病理性渗透混合物是许多致命疾病的一个促成因素。我们希望了解天然渗透剂混合物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，并阐明渗透剂在许多疾病中的作用。这样的信息将是非常有价值的，在确定通过药物，补充剂，或饮食的细胞的渗透调节剂含量的操纵目标。该项目意义重大，因为它克服了理解细胞病理生理学的一个主要障碍，即当渗透剂与蛋白质和代谢物在其天然混合物中结合时出现什么新特性的问题，从而开辟了改善疾病的新机会。这种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。 公共卫生相关性：该项目研究渗透剂（人体内自然存在的小分子）的混合物，以及这些混合物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剂，以对抗各种癌症和渗透剂发挥作用的其他疾病。

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.