The Molecular Basis of Cellular Control Mechanisms

描述(申请人提供)：这个项目的长期目标是阐明生物系统中调控信号传递的过程，作为了解代谢控制的分子机制的一种手段。了解细胞如何调节和控制其功能的各个方面，对于我们在这些控制机制崩溃时进行干预的能力至关重要。几乎所有的信号转导模式都可以以某种方式与蛋白质构象的变化相关。例如，胰岛素或胰高血糖素与其受体结合引起的构象变化调节血糖水平，或者变构酶的大的四级构象变化通过改变其催化活性来调节代谢途径。在这个项目期间，我们将集中研究涉及信号传输和变构控制的三个系统。天冬氨酸转氨甲酰酶(ATCase)和1，6-二磷酸果糖酶(FBPase)分别参与控制嘧啶和糖异生途径的速率。这两种酶都经历了戏剧性的构象变化，包括环路运动和整个蛋白质亚基的运动，以实现其功能。此外，我们还将研究合作酶--二氢罗丹明酶。在嘧啶核苷酸生物合成中，这种酶经历与催化活性相协调的环状运动。将使用各种方法从分子层面了解这些酶是如何发挥作用的。这个项目直接解决了生物信号如何传递的基本问题，特别是变构调节如何控制酶的活性。这一应用程序的具体目标分为基本部分和实际部分。我们将使用最先进的技术，包括时间分辨小角X射线散射和时间分辨X射线结晶学，在原子水平上捕捉信号分子对这些酶系统的影响的细节。我们还将在这些酶中使用战略性放置的荧光探针，既可以监测构象变化，也可以将这些构象变化与其功能联系起来。我们将使用X射线结晶学来定义ATCase的催化和变构机制中的每一步，包括在键形成的时刻。我们还将利用这些结构数据来设计高效的ATCase和FBPase抑制剂，这些抑制剂可能用于开发新的抗增殖、抗疟疾和抗糖尿病药物。了解这些蛋白质的构象变化和变构调控之间的关系也将有助于我们阐明细胞调控机制的分子基础。 对于控制大多数生物过程的调控信号传递的原子水平细节的理解，对于我们在这些控制失效时进行干预的能力至关重要。这项应用将集中在产生核酸前体的途径中所涉及的酶中调节信号的传递，以及参与维持血糖水平的途径之一。在分子水平上了解这些酶是如何控制它们各自的途径的，将为合理开发抗癌、抗糖尿病和抗疟疾新药提供基础。

DESCRIPTION (provided by applicant): The long term objectives of this project are to elucidate the processes involved in the transmission of regulatory signals in biological systems, as a means toward understanding the molecular mechanisms of metabolic control. The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of signal transduction can be related in some manner to protein conformational changes. For example, the conformational changes induced by the binding of insulin or glucagon to their receptors modulate blood sugar levels, or the large quaternary conformational changes of allosteric enzymes regulate metabolic pathways by altering their catalytic activity. During this project period we will concentrate on three systems involved in signal transmission and allosteric control. The allosteric enzymes aspartate transcarbamoylase (ATCase) and fructose 1,6- bisphosphatase (FBPase) are involved in controlling the rates of the pyrimidine and gluconeogenesis pathways, respectively. Both of these enzymes undergo dramatic conformational changes involving loop motions and movements of entire protein subunits for their function. In addition, we will study the cooperative enzyme, dihydroorotase. This enzyme in pyrimidine nucleotide biosynthesis undergoes loop motions that are coordinated with catalytic activity. A variety of approaches will be used to acquire a molecular-level understanding of how these enzymes function. This project directly addresses fundamental questions of how biological signals are transmitted, in general, and how allosteric regulation controls enzymatic activity, in particular. The specific aims of this application are divided into fundamental and practical components. We will use state of the art techniques involving time-resolved small-angle X-ray scattering and time-resolved X-ray crystallography to capture the details of the effects that signaling molecules have on these enzymatic systems at the atomic level. We will also use strategically placed fluorescent probes in these enzymes, both to monitor the conformational changes and to relate these conformational changes to their function. We will use X-ray crystallography to define each step in the catalytic and allosteric mechanisms of ATCase, including at the moment of bond formation. We will also use these structural data to design highly potent inhibitors of ATCase and FBPase that may be used for the development of new anti-proliferation, anti-malarial and anti-diabetic agents. Understanding of the relationship between conformational changes and allosteric control in these proteins will also help us elucidate the molecular basis of cellular control mechanisms. The understanding of the atomic level details of the transmission of regulatory signals, which control most biological processes, is critical for our ability to intervene when these controls break down. This application will concentrate on transmission of regulatory signals in enzymes involved in the pathway that produces the precursors of the nucleic acids, and of one of the pathways involved in maintaining blood sugar levels. A molecular level understanding of how these enzymes exert control over their respective pathway will provide a basis for the rational development of new anti-cancer, anti-diabetic and anti-malarial drugs.