Lipid electrophile adduction of Pin1 and effects on substrate binding & activity

Pin1 的脂质亲电子加合及其对底物结合的影响

• 批准号: 8472350
• 负责人: Christopher D Aluise
• 金额: $ 4.2万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-02-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8472350
• 关键词: 4 hydroxynonenal; Acrolein; Adverse effects; Aging; Aldehydes; Amino Acids; Binding; Carbohydrates; Cdc25C protein; Cells; Central Nervous System Diseases; Chemistry; DNA; Disease; Etiology; FOS gene; Goals; Human; In Vitro; Investigation; Large Intestine Carcinoma; Lipid Peroxidation; Lipids; Malignant Epithelial Cell; Malignant Neoplasms; Malondialdehyde; Maps; Membrane; Methods; Modification; NF-kappa B; Oxidative Stress; Peptidylprolyl Isomerase; Peripheral; Phosphorylation; Pin1 protein; Polyunsaturated Fatty Acids; Post-Translational Protein Processing; Predisposition; Process; Production; Proline; Protein Dephosphorylation; Protein phosphatase; Proteins; Proteomics; Protocols documentation; Reactive Oxygen Species; Research; Site; Structure; Tertiary Protein Structure; cis-trans-Isomerases; experience; human disease; insight; novel; overexpression; oxidation; oxidative damage; protein function; response

DESCRIPTION (provided by applicant): Oxidative damage to cellular lipids is a contributing factor to aging, as well as many peripheral and central nervous system diseases. Reactive oxygen species-induced modification of polyunsaturated fatty acids results in the production of electrophilic metabolites capable of irreversibly modifying proteins, carbohydrates, and DNA. Examples of lipid electrophiles that have been extensively studied are 4-hydroxynonenal (4-HNE), 4- oxononenal (4-ONE), acrolein, and malondialdehyde. Adduction by lipid electrophiles distorts protein tertiary structure and typically has adverse effects on protein function. Despite an undeniable connection to aging as well as a multitude of diseases where oxidative stress occurs, the susceptibility of peptidyl-prolyl cis/trans isomerase A1 (Pin1) to adduction by different electrophiles has not been thoroughly investigated. In human colorectal carcinoma cells (RKO), I will map the adduction sites of Pin1 in vitro using proteomics. I will examine the potential downstream effects of 4-HNE and 4-ONE adduction to Pin1 in terms of substrate binding and prolyl isomerization activity. Furthermore, I propose that Pin1 modification by electrophiles results in an inability to catalyze the proline-directed isomerization of several Raf1 moieties necessary for dephosphorylation by protein phosphatase 2A, resulting in prolonged hyperphosphorylation/inactivation. This investigation represents the first study to mechanistically examine electrophile adduction of Pin1 and the subsequent regulatory fate of its substrates. The proposed research allows for significant insight into the cellular response to Pin1 protein modification as a consequence of lipid peroxidation.

描述（由申请人提供）：细胞脂质的氧化损害是导致衰老的原因，以及许多周围神经系统疾病。活性氧诱导的多不饱和脂肪酸的修饰导致产生能够不可逆地修饰蛋白质，碳水化合物和DNA的亲电代谢产物。已经进行了广泛研究的脂质电力的实例是4-羟基苯烯（4-HNE），4-氧烯烯（4- ONE），丙烯醛和丙烯醛。脂质电力的内收蛋白会扭曲蛋白质的第三纪结构，通常对蛋白质功能产生不利影响。尽管与发生氧化应激的多种疾病以及多种疾病之间的联系，但尚未彻底研究肽基 - 丙酰基CIS/反式异构酶A1（PIN1）对内收内部的易感性。在人类结直肠癌细胞（RKO）中，我将使用蛋白质组学在体外绘制PIN1的内收位点。我将根据底物结合和脯氨酰同构活性来检查4-HNE和4对PIN1的潜在下游效应。此外，我提出，通过电生物的PIN1修饰导致无法催化蛋白质磷酸酶2a去磷酸化所必需的几种RAF1部分的脯氨酸定向异构化，从而导致延长的强磷酸化/灭活。这项研究代表了机械方面的首次研究 检查PIN1的亲电体收集及其底物的随后调节命运。拟议的研究允许由于脂质过氧化而对细胞对PIN1蛋白质修饰的细胞反应进行重大洞察力。