Functional Alterations in CDK2 by Lipid Electrophile Modification

脂质亲电子修饰对 CDK2 的功能改变

• 批准号: 8837744
• 负责人: Jeannie Camarillo
• 金额: $ 2.73万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-02 至 2016-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8837744
• 关键词: 4 hydroxynonenal; Affect; Amino Acids; Antioxidants; Apoptosis; Binding; Biological Assay; Cancer cell line; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Proteins; Cell physiology; Cells; Chemistry; Colorectal Cancer; Complex; Cyclin A; Cyclin E; Cyclins; Cycloheximide; DNA; Data; Development; Diffuse; Ensure; Epidemiologic Studies; Eukaryotic Cell; Exposure to; G1 Phase; G1/S Transition; G2 Phase; Generations; Genes; Genomic Instability; Genomics; Histidine; Human; Immunoblotting; In Vitro; Inflammation; Knowledge; Lead; Lipid Peroxidation; Lipid Peroxides; Lipids; Lysine; Malignant Neoplasms; Mammalian Cell; Membrane; Membrane Lipids; Methods; Modification; Oxidative Stress; Pathway interactions; Phase; Phosphotransferases; Play; Polyunsaturated Fatty Acids; Post-Translational Protein Processing; Process; Production; Protein Biosynthesis; Protein Kinase; Proteins; Proteomics; Reaction; Reactive Oxygen Species; Recombinants; Relative (related person); Research; Resistance; Role; S Phase; Side; Site; Structure-Activity Relationship; Testing; cell growth; epigenetic marker; gene repression; genetic regulatory protein; human CDK2 protein; in vitro Assay; mitochondrial membrane; mutant; polyunsaturated fat; protein degradation; protein function; protein protein interaction; protein structure; public health relevance; research study; response; tandem mass spectrometry; toxicant; tumorigenesis

 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.

 描述（由申请人提供）：实验和流行病学研究均表明，持续氧化应激导致的铬性炎症是癌症发展的一个促成因素。炎症可以通过许多不同的途径促进肿瘤发生，包括促进基因组不稳定性、表观遗传标记物的改变、增殖增加和对细胞凋亡的抵抗。虽然活性氧物质（ROS）单独能够诱导这些不同的途径，以促进转化，ROS的高反应性导致在生产位点附近的反应。膜双层内的多不饱和脂质，尤其是线粒体膜，是ROS的关键靶点。这些脂质与ROS之间的反应导致脂质过氧化，并且在某些情况下，可以产生小的反应性脂质亲电体。4-羟基壬烯醛（HNE）是最常研究的脂质亲电体。由于其相对于ROS的较低反应性，HNE可以在整个细胞中扩散并共价修饰DNA和蛋白质;细胞蛋白质的修饰可以导致功能改变。先前的蛋白质组学数据已经显示了许多易于被HNE修饰的蛋白质，其中之一是CDK 2。细胞周期蛋白依赖性激酶2（CDK 2）是一种细胞周期蛋白，负责G1/S转换和维持S期进展。有趣的是，微阵列研究显示，HNE治疗后与S期相关的基因下调;这些基因中的许多基因在上游受CDK 2激酶活性控制。总之，这些数据表明，CDK 2的HNE修饰可以改变其功能，导致蛋白质失活和细胞周期停滞。我们的初步数据表明，CDK 2被HNE修饰在许多组氨酸和赖氨酸残基上。用HNE处理同步化的细胞导致进展到S期的延迟。最值得注意的是，用HNE处理的细胞的处理导致CDK 2激酶活性降低。鉴于这些数据，我们假设HNE共价修饰CDK 2并抑制其激酶活性，从而导致G1期阻滞，直到HNE修饰的CDK 2被降解。我提出了两个目的来检验这一假设：1）评估HNE修饰CDK 2的功能影响和2）量化RKO细胞中HNE修饰CDK 2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修饰导致的蛋白质功能改变的理解。提出的目标将提供一种机制，说明HNE加合引起的蛋白质结构的微小变化如何显著破坏细胞内的相互作用。