CHARACTERIZATION OF THE EARLY APOPTOTIC MITOCHONDRIAL PROTEOME

早期凋亡线粒体蛋白质组的表征

• 批准号: 8363803
• 负责人: ALMA L BURLINGAME
• 金额: $ 0.01万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363803
• 关键词: Address; Affinity; Apoptosis; Apoptotic; Bioinformatics; Biology; Cell Death; Chemicals; Electrophoresis; Evolution; Funding; Goals; Grant; Histocompatibility Testing; Label; Mass Spectrum Analysis; Mitochondria; Mitochondrial Proteins; Modeling; N-terminal; National Center for Research Resources; Nuclear; Organelles; Pathway interactions; Peptide Signal Sequences; Peptides; Preparation; Principal Investigator; Proteins; Proteome; Proteomics; Relative (related person); Research; Research Infrastructure; Resources; Ribosomes; Role; Sampling; Signal Transduction; Source; United States National Institutes of Health; Yeasts; base; cost; mitochondrial membrane; novel; protein aminoacid sequence; subtiligase

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. Several proteomic studies of mammalian and yeast mitochondria have identified a growing list of over 1000 proteins in the mitochondrial proteome. The overlap between various studies is limited to 60-80% and the relative abundance of these components varies with tissue type. The number of mitochondrial proteins has been estimated at 2000, based on an endosymbiont model for mitochondrial evolution (Gabaldon, 2004), thus the mitochondrial proteome is still incomplete. The vast majority of mitochondrial proteins are nuclear encoded and are expected to contain N-terminal peptide signals used for mitochondrial targeting. Interestingly, only one third of the proteins in the mammalian mitochondrial proteome are predicted to contain a signal peptide motif using bioinformatic analysis, while another third are easily annotated with known mitochondrial enzymatic functions. The remaining third of the sample are typically novel mitochondrial localized proteins of ambiguous function, and in some studies this number is greater than half of the total protein list. Conspicuously absent from many of these protein lists are the apoptosis regulating proteins, which associate with mitochondrial membranes in a highly regulated pathway for cell death. This project will address 1) functional annotation of mitochondrial proteins using controlled biology and pharmacological induction of early apoptosis signaling and 2) analysis of N-terminal peptide for targeting of proteins to mitochondria. To accomplish these goals, we are investigating free flow electrophoresis (FFE) purification of intact mitochondria, reducing contamination by high abundance ribosome and nuclear components. We are also using a chemical biology enzymatic approach to label with subtiligase the free N-termini of mitochondrial proteins for affinity capture. The role of the Mass Spectrometry Facility in this project is to handle all aspects of the organelle preparation, peptide sequencing and bioinformatic analysis of N-terminal peptides.

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。 几项对哺乳动物和酵母线粒体的蛋白质组学研究发现，线粒体蛋白质组中有1000多种蛋白质。不同研究之间的重叠被限制在60%-80%，这些成分的相对丰度因组织类型而异。根据线粒体进化的内共生模型(Gabaldon，2004)，线粒体蛋白质的数量估计为2000个，因此线粒体蛋白质组仍然不完整。绝大多数线粒体蛋白是核编码的，预计含有用于线粒体靶向的N末端多肽信号。有趣的是，通过生物信息学分析预测，哺乳动物线粒体蛋白质组中只有三分之一的蛋白质含有信号肽基序，而另三分之一的蛋白质很容易用已知的线粒体酶功能进行注释。样本中剩下的三分之一是典型的功能不明确的新型线粒体定位蛋白，在一些研究中，这个数字超过了总蛋白清单的一半。值得注意的是，许多这些蛋白质清单中没有凋亡调节蛋白，它们与线粒体膜有关，在细胞死亡的高度调控途径中。 该项目将涉及1)利用早期凋亡信号的受控生物学和药理学诱导对线粒体蛋白质进行功能注释，以及2)分析N末端多肽以将蛋白质靶向线粒体。为了实现这些目标，我们正在研究自由流动电泳法(FFE)纯化完整的线粒体，减少高丰度核糖体和核成分的污染。我们还使用了一种化学生物酶方法，用枯草杆菌酶标记线粒体蛋白质的游离N-末端，以获得亲和力。质谱仪在该项目中的作用是处理N端肽的细胞器制备、肽序列和生物信息学分析的所有方面。