SIGNALING CASCADES, ALLOSTERY AND THE PYRIMIDINE PATHWAY

信号级联、变构和嘧啶途径

• 批准号: 6498709
• 负责人: HEDEEL I. GUY EVANS
• 金额: $ 19.22万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498709
• 关键词: allosteric site; amidohydrolases; aspartate carbamoyltransferase; biological signal transduction; carbamoylphosphate synthase; enzyme activity; enzyme mechanism; mitogen activated protein kinase; orotate; phosphorylation; protein kinase A; purine /pyrimidine metabolism; pyrophosphatase; tissue /cell culture; transfection; uridine triphosphate

In mammalian cells, the rate of de novo pyrimidine biosynthesis is regulated by the multi-functional protein CAD. The protein, which catalyzes the first half of the pathway, consists of six copies of a 243 kDa polypeptide folded into separate structural domains that carry glutamine dependent carbamoyl phosphate synthetase (CPSase), aspartate transcarbomoylase (ATCase) and dihydrooortase (DHOase) activities. The activity of the pathway is precisely controlled and increases when cells are induced to proliferate. Studies of the purified protein have shown that CPSase, which catalyzes the initial, rate limiting step in the pathway is the major locus of regulation and is allosterically controlled by UTP, an inhibitor and PRPP, an activator. In addition, the activity of the complex is regulated by protein kinase A mediated phosphorylation, although its role in regulating the growth state and cell cycle changes in the activity of the pyrimidine biosynthetic pathway is not at all clear. The recent discovery that EGF stimulation results in MAP kinase mediated phosphorylation and activation of CAD makes its possible to resolve this ambiguity. The objective of this research is to decipher the interrelationships and assess the importance of all these control mechanisms on the regulation of pyrimidine biosynthesis in vivo. The rate of de no pyrimidine biosynthesis, the size of the allosteric effector pools and the phosphorylation state of the protein will be examined in mammalian cells grown in culture and in transfectants that express CAD mutants in which one or more of the regulatory mechanisms have been disabled. The approach will be to extrapolate the extensive information developed on the regulation of purified CAD to develop a comprehensive model that can account for the in vivo regulation of de novo pyrimidine biosynthesis in different growth states, phases of the cycle and in cells stimulated by growth factors.

在哺乳动物细胞中，从头嘧啶生物合成的速率由多功能蛋白CAD调节。该蛋白催化途径的前半部分，由折叠成单独结构域的243 kDa多肽的六个拷贝组成，所述结构域携带谷氨酰胺依赖性氨甲酰磷酸合成酶（CPS酶）、天冬氨酸转羧酰酶（ATCase）和二氢乳清酸酶（DHOase）活性。该途径的活性受到精确控制，并在细胞被诱导增殖时增加。对纯化蛋白的研究表明，催化该途径中的初始限速步骤的CPS酶是主要的调节位点，并且受UTP（抑制剂）和PRPP（激活剂）的变构控制。此外，该复合物的活性由蛋白激酶A介导的磷酸化调节，尽管其在调节嘧啶生物合成途径活性的生长状态和细胞周期变化中的作用尚不清楚。最近发现EGF刺激导致MAP激酶介导的CAD磷酸化和激活，这使得解决这一模糊性成为可能。本研究的目的是破译的相互关系，并评估所有这些控制机制的重要性，在体内嘧啶生物合成的调节。脱氧嘧啶生物合成的速率、变构效应物库的大小和蛋白质的磷酸化状态将在培养中生长的哺乳动物细胞和表达CAD突变体的转染子中进行检查，其中CAD突变体中的一种或多种调节机制已被禁用。该方法将是外推广泛的信息开发的纯化CAD的调节，以开发一个全面的模型，可以占从头嘧啶生物合成在不同的生长状态，周期的阶段，并在生长因子刺激的细胞中的体内调节。