Functional Alterations in CDK2 by Lipid Electrophile Modification

 描述(由申请人提供)：实验和流行病学研究都表明，由于持续的氧化应激，慢性炎症是癌症发展的一个促成因素。炎症可通过许多不同的途径促进肿瘤的发生，包括促进基因组的不稳定、表观遗传标记的改变、增殖的增加和对凋亡的抵抗。虽然活性氧物种(ROS)本身能够诱导这些不同的途径来促进转化，但ROS的高反应性导致在产生地点附近发生反应。膜双层内的多不饱和脂类，尤其是线粒体膜，是ROS的关键靶标。这些脂质和ROS之间的反应会导致脂质过氧化，在某些情况下，会产生小的、活性的脂类亲电体。4-羟基壬烯醛(HNE)是目前研究最多的一种脂质亲和剂。由于相对于ROS，HNE的反应性较低，可以扩散到整个细胞，并以共价方式修饰DNA和蛋白质；细胞蛋白质的修饰可以导致功能改变。以前的蛋白质组学数据显示，许多蛋白质容易被HNE修饰，其中之一是CDK2。细胞周期蛋白依赖性蛋白2(CDK2)是一种细胞周期蛋白，参与细胞周期蛋白G1/S的转换和维持细胞周期通过S时相的进程。有趣的是，微阵列研究显示，在HNE治疗后，与S期相关的基因下调；其中许多基因上游受CDK2激酶活性控制。综上所述，这些数据表明，CDK2的HNE修饰可能改变其功能，导致蛋白质失活和细胞周期停滞。我们的初步数据表明，CDK2被HNE修饰在一些组氨酸和赖氨酸残基上。用HNE处理同步化细胞会导致进入S期的延迟。最值得注意的是，用HNE处理的细胞导致CDK2激酶活性下降。有了这些数据，我们假设HNE共价修饰CDK2并抑制其激酶活性，从而导致G1期停滞，直到HNE修饰的CDK2被降解。我提出了两个目的来验证这一假说：1)评估HNE修饰CDK2的功能影响；2)量化HNE在RKO细胞中修饰CDK2的程度。本申请中提出的研究将评估HNE修饰对主要细胞周期调节因子和激酶的功能意义，从而增加我们对HNE修饰引起的蛋白质功能变化的理解。提出的目标将提供一种机制，说明HNE对蛋白质结构的微小变化如何显著扰乱细胞内的相互作用。

 DESCRIPTION (provided by applicant): Chromic inflammation, as a result of continuous oxidative stress, has been shown to be a contributing factor to the development of cancer by both experimental and epidemiological studies. Inflammation can contribute to tumorigenesis by a number of different pathways, including promotion of genomic instability, alterations of epigenetic markers, increases in proliferation, and resistance to apoptosis. While reactive oxygen species (ROS) alone are capable of inducing these different pathways to promote transformation, the high reactivity of ROS results in reactions near the site of production. Polyunsaturated lipids within membrane bilayers, most notably mitochondrial membranes, are key targets of ROS. Reactions between these lipids and ROS result in lipid peroxidation and, in some cases, can generate small, reactive lipid electrophiles. 4-Hydroxynonenal (HNE) is the most commonly studied lipid electrophile. Due to its lower reactivity relative to ROS, HNE can diffuse throughout the cell and covalently modify both DNA and proteins; modification of cellular proteins can result in altered function. Previous proteomic data have shown a number of proteins susceptible to modification by HNE, one of which is CDK2. Cyclin-dependent kinase 2 (CDK2) is a cell cycle protein responsible for the G1/S transition and for maintaining progression through S-phase. Interestingly, microarray studies have shown down-regulation of genes associated with S-phase following HNE treatment; many of these genes are controlled upstream by CDK2 kinase activity. Together, these data suggest that HNE-modification of CDK2 could alter its function, resulting in protein inactivation and cell cycle arrest. Our preliminary data hve shown that CDK2 is modified by HNE on a number of histidine and lysine residues. Treatment of synchronized cells with HNE causes a delay in progression into S-phase. Most notably, treatment of cells treated with HNE results in a decrease in CDK2 kinase activity. Given these data, we hypothesize that HNE covalently modifies CDK2 and inhibits its kinase activity, thereby resulting in G1-phase arrest until HNE-modified CDK2 is degraded. I propose two aims to test this hypothesis: 1) Assess the functional impact of HNE modification of CDK2 and 2) Quantify the extent of CDK2 modification by HNE in RKO cells. The research proposed in this application will assess the functional significance of HNE modification on a main cell cycle regulator and kinase, thereby increasing our understanding of altered protein function as a result of HNE modification. The aims proposed will provide a mechanism for how small changes in protein structure from the adduction by HNE can significantly disrupt interactions within the cell.