MODE OF ACTION OF A BACTERIAL FUNCTION INVOLVED IN CELL GROWTH CONTROL

参与细胞生长控制的细菌功能的作用方式

• 批准号: 3963039
• 负责人: S GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3963039
• 关键词: DNA repair; Escherichia coli; bacterial genetics; bacterial polysaccharides; cell capsule; cell growth regulation; genetic manipulation; genetic mapping; genetic regulation; lac operon; molecular biology; mucopolysaccharides; mutant; operon; peptidases; pleiotropism; proteolysis; regulatory gene

We have been studying the role that protein degradation plays in regulating cell growth control, through the study of mutants defective in ATP-dependent protein degradation. E. coli lon mutants are defective in cell division regulation after DNA damage, and we have previously demonstrated that this defect is due to stabilization of a highly unstable cells division inhibitor, the product of the SulA gene. lon mutants also overproduce capsular polysaccharide; we have identified three regulatory genes which participate in the regulation of cps (capsule synthesis) genes. The protein products of two positive regulatory loci, rcsA and rcsB, have been identified. RcsA is unstable, and its turnover may be mediated by the Lon protease; RcsB is stable. The third regulatory locus, rcsC, serves as a negative regulator of the system, but may have additional roles as well. We have used the in vitro degradation of the Lon substrates lambda N protein, insulin B chain, and glucagon, to define the sites of Lon cleavage. While Lon has preferred sites of cleavage on these molecules, the preferred sites do not represent any simple amino acid sequence. Studies on cells devoid of Lon activity demonstrate the existence of other ATP-dependent proteolysis systems in E. coli. A genetic selection for mutants with increased activity for other proteases has been developed, by selecting for mutants resistant to the unstable cell division inhibitor, SulA. Mutant candidates which are resistant to high levels of SulA apparently increase proteolysis. Mapping studies, now in progress, should allow the identification of the proteases involved. Using a biochemical screen for the ATP-dependent degradation of casein, we have identified a new, multi-component ATP-dependent protease in cells devoid of Lon activity.

我们一直在研究蛋白质降解在调节 通过对缺陷突变株的研究来控制细胞生长 依赖于ATP的蛋白质降解。大肠埃希氏菌离子突变体在 DNA损伤后的细胞分裂调控，我们之前已经 证明这一缺陷是由于高度不稳定的 细胞分裂抑制因子，是苏拉基因的产物。Lon变种人也 过量生产荚膜多糖；我们已经确定了三种调节 参与囊膜合成调控的基因 基因。RCSA和RCSA两个正调控基因座的蛋白质产物 RCSB，已被识别。RCSA不稳定，其营业额可能会 由Lon蛋白酶介导；RCSB是稳定的。第三个调控点， RCSC，作为系统的负调节器，但可能有额外的 角色也是如此。我们使用了Lon底物的体外降解 Lambda N蛋白、胰岛素B链和胰升糖素，以确定Lon的位置 乳沟。虽然Lon在这些分子上有首选的裂解部位， 首选位点不代表任何简单的氨基酸序列。 对缺乏Lon活性的细胞的研究证明了其他 大肠杆菌中依赖三磷酸腺苷的蛋白分解系统。一种遗传选择 已经开发出对其他蛋白酶具有更高活性的突变体，通过 筛选对不稳定细胞分裂抑制物具有抗性的突变体， 秀拉。对高水平秀拉耐药的突变体候选 明显增加了蛋白质的分解。目前正在进行的测绘研究应该 允许鉴定所涉及的蛋白酶。使用一种生物化学 筛选酪蛋白依赖于ATP的降解，我们已经确定了一种 无Lon活性的细胞中新的多组分ATP依赖的蛋白水解酶。