Investigating cysteine-mediated protein activities in C. elegans

研究线虫中半胱氨酸介导的蛋白质活性

• 批准号: 9119183
• 负责人: Eranthie Weerapana
• 金额: $ 36.43万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-03 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9119183
• 关键词: Accounting; Address; Affect; Age of Onset; Aging; Aging-Related Process; Animal Model; Binding Proteins; Biological Models; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Characteristics; Chemicals; Complement; Cysteine; Data; Degenerative Disorder; Development; Disease; Event; Gene Expression; Genetic; Genetic study; Genomics; Health; Homologous Gene; Housing; Human; In Situ; In Vitro; Insulin; Insulin-Like Growth Factor I; Label; Life; Life Extension; Link; Lipid Binding; Lipids; Longevity; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Modification; Molecular; Mutation; Nematoda; Organism; Orthologous Gene; Oxidation-Reduction; Oxidative Stress; Phenotype; Post-Translational Protein Processing; Predisposition; Proteins; Proteolysis; Proteomics; RNA Interference; Reactive Oxygen Species; Regulation; Reporting; Research Design; Resistance; Role; Signal Pathway; Signal Transduction; Site; Stress; Sulfhydryl Compounds; Technology; Testing; Time; age related; base; cytosolic lipid-binding proteins; in vivo; inhibitor/antagonist; insight; insulin mediators; interest; knock-down; lipid metabolism; mutant; novel; oxidation; photolysis; receptor; small molecule; small molecule libraries; therapeutic development; therapeutic target; tool; transcription factor; transcriptomics

 DESCRIPTION (provided by applicant): The molecular mechanisms underlying the aging process are poorly understood, thereby hindering the development of therapeutics to delay the onset of aging and age-related degenerative diseases. Genetic studies in the widely used model organism, Caenorhabditis elegans, have generated daf-2 mutants defective in insulin/IGF-1 signaling (IIS), which have significantly extended lifespans. The daf-2 mutation activates DAF-16, a transcription factor, which initiates downstream gene expression changes that mediate the life-extension phenotype. We have identified protein-activity changes that are downstream effects of DAF-16 activation using the tools of chemical proteomics. These studies complement conventional genomic and proteomic approaches by providing insight into low-abundance proteins and posttranslational modifications (PTMs) implicated in IIS. From our preliminary studies, we identified a lipid-binding protein, LBP-3, which upon RNAi-mediated knockdown increases both lifespan and dauer formation in C. elegans. Given the established dysregulation of lipid metabolism in IIS, and the confirmed role of other lipid-binding proteins in controlling lifespan and stress resistance, we hypothesize that LBP-3 is a novel mediator of IIS. To test this hypothesis, we will determine the mechanism by which LBP-3 acts within known nodes of the IIS pathway to regulate lifespan. Furthermore, since mammalian homologs of LBP-3 are known to be redox regulated, and dysregulation of reactive oxygen species (ROS) levels is a characteristic feature of IIS, we will investigate the in vivo oxidation state of LBP-3. In additio to revealing a novel mode of regulation for C. elegans LBP-3, these studies will also serve to more globally evaluate protein oxidation events accompanying IIS. Lastly, since oxidation of mammalian LBPs is known to affect protein stability and lipid binding, we will evaluate the effect of oxidation on C. elegans LBP-3 function. LBP-3 contains a highly reactive cysteine residue, Cys154, which is the predicted site of oxidation. We will exploit this reactive cysteine to develop covalent small-molecule probes and inhibitors to pharmacologically modulate LBP-3 function in C. elegans. Together, these studies will: (1) characterize a novel downstream mediator of C. elegans IIS; (2) reveal the role of protein oxidation in governing the function of LBP-3 and other C. elegans proteins during IIS; and (3) demonstrate that LBP-3 can be targeted by small molecules to pharmacologically modulate C. elegans lifespan.

 描述（由申请人提供）：对衰老过程的分子机制知之甚少，从而阻碍了延缓衰老和与年龄相关的退行性疾病发生的治疗方法的开发。对广泛使用的模型生物体——秀丽隐杆线虫进行了遗传学研究。产生了胰岛素/IGF-1信号传导（IIS）缺陷的daf-2突变体，该突变体显着延长了寿命。 daf-2突变激活了转录因子DAF-16，从而显着延长了寿命。启动介导寿命延长表型的下游基因表达变化我们使用化学蛋白质组学工具确定了 DAF-16 激活的下游影响的蛋白质活性变化，通过提供对低表达的见解来补充传统的基因组和蛋白质组学方法。 IIS 中涉及的蛋白质丰度和翻译后修饰 (PTM) 从我们的初步研究中，我们发现了一种脂质结合蛋白 LBP-3，在 RNAi 介导的敲低后，它可以延长寿命并延长寿命。鉴于 IIS 中脂质代谢的失调以及其他脂质结合蛋白在控制寿命和抗应激性中的作用得到证实，我们发现 LBP-3 是 IIS 的一种新型介质，以对此进行测试。假设，我们将确定 LBP-3 在 IIS 途径的已知节点内发挥作用以调节寿命的机制。此外，由于已知 LBP-3 的哺乳动物同源物受到氧化还原调节和反应性调节失调。氧物种（ROS）水平是IIS的一个特征，我们将研究LBP-3的体内氧化态，除了揭示秀丽隐杆线虫LBP-3的新调节模式外，这些研究还将有助于更多的研究。最后，由于已知哺乳动物 LBP 的氧化会影响蛋白质稳定性和脂质结合，因此我们将评估氧化对秀丽隐杆线虫 LBP-3 的影响。 LBP-3含有一个高反应性半胱氨酸残基Cys154，这是我们将利用这种反应性半胱氨酸来开发的预测位点。 共价小分子探针和抑制剂可在秀丽隐杆线虫中药理学调节 LBP-3 功能，这些研究将：(1) 表征秀丽隐杆线虫 IIS 的新型下游介质；(2) 揭示蛋白质氧化在调控中的作用。 LBP-3 和其他秀丽隐杆线虫蛋白在 IIS 过程中的功能；以及 (3) 证明 LBP-3 可以被小分子靶向以药理调节秀丽隐杆线虫寿命。