CHARACTERIZATION OF SMALL MOLECULE METABOLITES

小分子代谢物的表征

• 批准号: 8365852
• 负责人: STANLEY FIELDS
• 金额: $ 2.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365852
• 关键词: Amines; Anabolism; Anemia; Arginine; Biology; Capillary Electrophoresis; Cells; Chemicals; Complex; Disease; Doxycycline; Essential Genes; Exhibits; Exposure to; Funding; Fungal Genome; Gene Cluster; Gene Deletion; Genes; Grant; Human; Lead; Lysine; Mass Spectrum Analysis; Metabolism; Methods; National Center for Research Resources; Nutrient; Pathway interactions; Pharmaceutical Preparations; Principal Investigator; Process; RPS19 gene; Relative (related person); Research; Research Infrastructure; Resources; Ribosomal Proteins; Saccharomyces cerevisiae; Sampling; Source; United States National Institutes of Health; Vitamins; Work; Yeasts; base; cofactor; cost; human disease; interest; mutant; new technology; small molecule

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. Metabolism encompasses all the processes by which a cell generates energy and other essential molecules from nutrients. These pathways rely on hundreds of genes and involve thousands of small molecule intermediates, vitamins and cofactors. To characterize small molecule metabolites in the yeast Saccharomyces cerevisiae, we used chemical derivatization in combination with capillary electrophoresis. We quantified primary amine-containing metabolites in ~4500 yeast strains, each lacking a single non-essential gene, and clustered the strains based on the similarities among profiles to identify genes involved in related processes. Using this approach we found that strains lacking genes involved in arginine biosynthesis all show a similar accumulation of lysine, demonstrating that the clustering approach allows related genes to be assigned to pathways. In addition to the clustering approach, we identified a number of strains exhibiting unique and interesting profiles. Many of the genes identified in these analyses have not been previously characterized; others encode components of well-known complexes. This approach has already yielded a few interesting discoveries relevant to human disease. We observed a dramatic accumulation of an unknown metabolite in strains lacking the gene RPS19, and found that deletion of the genes for many ribosomal proteins results in accumulation of the same metabolite. Haploinsufficiency of RPS19 in humans leads to Blackfan?s anemia, while loss of other ribosomal proteins can lead to other varieties of anemia. The identification of the metabolite accumulation in the rps19 mutant may allow better understanding of this disease in humans. Continuing work will focus on profiling yeast strains in each of which, one of 1000 of the essential genes can be turned off after exposure to the drug doxycycline. As a complementary approach, these strains will also be profiled using a new technology, GCxGC-TOFMS. This method has been shown to allow identification and relative quantification of several hundred metabolites in each sample. Mass spectrometry will also be used to further characterize some of the interesting mutant strains identified using the capillary electrophoresis.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 新陈代谢包括细胞从营养物质产生能量和其他必需分子的所有过程。 这些途径依赖于数百个基因，并涉及数千个小分子中间体，维生素和辅因子。 为了表征酿酒酵母中的小分子代谢产物，我们使用化学衍生结合毛细管电泳。我们定量了约4500种酵母菌株中含伯胺的代谢物，每种酵母菌株都缺乏一个非必需基因，并根据图谱之间的相似性对菌株进行聚类，以确定参与相关过程的基因。 使用这种方法，我们发现缺乏精氨酸生物合成相关基因的菌株都显示出相似的赖氨酸积累，表明聚类方法允许将相关基因分配给途径。 除了聚类方法，我们确定了一些菌株表现出独特的和有趣的配置文件。 在这些分析中发现的许多基因以前没有被鉴定过;其他基因编码众所周知的复合物的组分。 这种方法已经产生了一些与人类疾病有关的有趣发现。 我们观察到一种未知的代谢物在缺乏基因RPS 19的菌株中大量积累，并发现许多核糖体蛋白基因的缺失导致相同代谢物的积累。 人类RPS 19的单倍不足导致Blackfan？S贫血，而其他核糖体蛋白质的丢失可导致其他种类的贫血。在rps 19突变体中代谢物积累的鉴定可以更好地理解人类的这种疾病。 继续的工作将集中在分析酵母菌株中的每一个，1000个必需基因中的一个可以在暴露于药物强力霉素后被关闭。 作为补充方法，这些菌株也将使用新技术GCxGC-TOFMS进行分析。 该方法已被证明可以鉴别和相对定量每个样品中的数百种代谢物。 质谱法也将用于进一步表征使用毛细管电泳鉴定的一些有趣的突变株。