TURNOVER RATES IN THE PROTEIN POOL OF C ELEGANS WITH EXTREME LIFESPAN

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 线虫具有寿命短、遗传适应性好、可获得长寿突变株等优点，已成为衰老研究中最具吸引力的模型系统之一。在之前的一次合作中，我们在蠕虫中发现了非常有趣的蛋白质组变化，这些变化是由于饮食限制或胰岛素/IGF样信号的敲除而导致的。这些变化的一个重要部分与蛋白质周转有关。我们假设线虫寿命的延长是由特定蛋白质池周转的变化支持的。 在拟议的项目中，我们将应用敏感而可靠的方法脉冲SILAC，以验证与相应的对照相比，对躯体维持至关重要的蛋白质在长寿蠕虫中是否更快地被翻转。另一方面，我们预计这些长寿动物体内其他大部分蛋白质的周转率会出现补偿性下降。 在实验装置中，我们将使用两个寿命延长的突变株：DAF-2(胰岛素/IGF受体突变体)和IFE-2(体细胞翻译启动突变体)，以及一个通过环境操纵和通过食物稀释限制饮食来延长寿命的菌株。这三个寿命较长的菌株将与正常寿命的相应对照菌株进行比较。 蠕虫培养、SILAC标记和采样将在Braeckman实验室进行，该实验室以其在线虫培养和老化研究方面的丰富经验而闻名。这些样品随后将在PNNL使用最先进的世界级技术进行分析和量化，使用的是高分辨率和高质量精度的质谱学技术。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. With its short lifespan, genetic amenability and the availability of long-lived mutant strains, C. elegans has become one of the most attractive model systems for aging research. In a previous collaboration, we have discovered very intriguing proteomic changes in worms that are long-lived due to dietary restriction or knockdown of insulin/IGF-like signaling. An important part of these changes is related to protein turnover. We hypothesize that lifespan extension in C. elegans is supported by changes in the turnover of specific protein pools. In the proposed project, we will apply the sensitive and reliable method pulsed SILAC, to verify whether proteins that are important for somatic maintenance are turned over more rapidly in long-lived worms compared to the corresponding controls. On the other hand, we expect a compensatory decrease in the turnover of the bulk of other proteins in these long-lived animals. For the experimental setup, we will use two mutant strains with extended lifespan: daf-2 (which is an insulin/IGF receptor mutant) and ife-2 (a somatic translation initiation mutant) and one strain in which lifespan is extended by environmental manipulation and dietary restriction by food dilution. These three long-lived strains will be compared to a corresponding control strain with normal lifespan. Worm culturing, SILAC labeling and sampling will be performed in the Braeckman lab, known for its extensive experience with C. elegans culturing and aging studies. These samples will be subsequently analyzed and quantified with state-of-the-art world class technology utilizing high resolution and mass accuracy mass spectrometry technologies at PNNL.