Investigating cysteine-mediated protein activities in C. elegans

 描述(申请人提供)：衰老过程背后的分子机制知之甚少，因此阻碍了延缓衰老和与年龄相关的退行性疾病的治疗方法的发展。在广泛使用的模式生物秀丽线虫的遗传学研究中，已经产生了胰岛素/胰岛素样生长因子-1信号(IIS)缺陷的daf-2突变体，这些突变体显著延长了寿命。Daf-2突变激活了转录因子DAF-16，从而启动了下游基因表达的变化，从而介导了寿命延长的表型。我们已经利用化学蛋白质组学的工具确定了DAF-16激活的下游影响的蛋白质活性变化。这些研究通过深入了解IIS中涉及的低丰度蛋白质和翻译后修饰(PTM)，补充了传统的基因组和蛋白质组方法。从我们的初步研究中，我们确定了一种脂结合蛋白LBP-3，它在RNAi介导的击倒后延长了线虫的寿命和Dauer的形成。鉴于IIS中已证实的脂代谢失调，以及其他脂结合蛋白在控制寿命和应激抗性方面的已证实作用，我们假设LBP-3是IIS的一个新的介体。为了验证这一假说，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LBP-3的哺乳动物同源物是已知的氧化还原调节的，而活性氧物种(ROS)水平的失调是IIS的特征，因此我们将研究LBP-3在体内的氧化状态。除了揭示线虫LBP-3的一种新的调控模式外，这些研究还将有助于更全面地评估伴随IIS的蛋白质氧化事件。最后，由于哺乳动物LBPS的氧化已知会影响蛋白质的稳定性和脂质结合，我们将评估氧化对线虫LBP-3功能的影响。LBP-3含有一个高活性的半胱氨酸残基，Cys154，这是预测的氧化位点。我们将利用这种反应性半胱氨酸来开发 药物调节线虫LBP-3功能的共价小分子探针和抑制剂。综上所述，这些研究将：(1)表征线虫IIS的一个新的下游介体；(2)揭示在IIS过程中蛋白质氧化在调节LBP-3和其他线虫蛋白功能中的作用；以及(3)证明LBP-3可以被小分子靶向，从药物上调节线虫的寿命。

 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.