Poxvirus-mediated change to proteasomal peptide signatures in macrophages

痘病毒介导的巨噬细胞中蛋白酶体肽特征的变化

• 批准号: 8256460
• 负责人: DOROTA SKOWYRA
• 金额: $ 6.2万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8256460
• 关键词: 26S proteasome; Active Sites; Address; Affinity; Antibodies; Antiviral Agents; Area; Bioinformatics; Biological Assay; Biological Factors; Bone Marrow; Cell division; Cell physiology; Cells; Classification; Complex; Crude Extracts; Custom; Data; Disease model; Ectromelia; Electron Microscopy; Fellowship; Future; Gene Expression; Genes; Goals; Host Defense; Immune; In Vitro; Individual; Infection; Institutes; Interferons; Lead; Length; Mass Spectrum Analysis; Mediating; Modeling; Monitor; Mus; Organism; Outcome; Pathogenicity; Pathway interactions; Peptides; Poxviridae; Precipitation; Preparation; Procedures; Property; Proteins; Proteolysis; Recruitment Activity; Research; Role; Saccharomyces cerevisiae; System; Systems Biology; Technology; Testing; Training; Ubiquitin; Ubiquitin-mediated Proteolysis Pathway; Viral; Virus; Western Blotting; Work; Yeasts; base; bone; career; cell growth; cytochemistry; immunogenic; in vivo; interest; macrophage; multicatalytic endopeptidase complex; mutant; particle; skills; success; virology

DESCRIPTION (provided by applicant): This fellowship will revitalize the applicant's research career by stimulating a major change in the direction of her research, broadening her scientific background, and acquiring new skills and capabilities in the area of Virology. Our past work has focused on the mechanism by which ubiquitin-proteasome system regulates cell growth and division of the non-infectious, single-cell organism Saccharomyces cerevisiae. Of special interest is the finding that substrates can be trapped at the 26S proteasome in a manner that allows their later proteolysis under controlled in vitro conditions, thereby leading to an accumulation of peptides that can be analyzed by mass spectrometry. Our unpublished data suggest that this strategy could be used to monitor large pools of natural product peptides generated by mammalian 26S proteasome and its immune 26Si version stimulated by interferons (IFNs) that produces antigenic peptides. When applied to virus-infected cells, this analysis may reveal a specific "peptide signature" of virus-mediated changes in proteasomal proteolysis, including viral subversion of specific cellular pathways and natural precursors of antigenic peptides. These themes have a major significance in virology, but addressing them via traditional approaches has a low rate of success. We will characterize changes in pools of peptides generated from natural substrates co-purified with 26S or 26Si proteasomes from uninfected primary mouse macrophages and from macrophages infected with the poxvirus ectromelia (EV). EV overcomes the IFNs-mediated antiviral defenses of macrophages, which is key to its pathogenicity and may depend on a yet undefined form of proteasome. In Aim 1, we will characterize the 26S and 26Si proteasomes expressed in uninfected and EV-infected macrophages in the presence or absence of IFNs. We will use qPCR, quantitative western blot and immuno-cytochemistry to detect expression of 26S and 26Si specific subunits; immuno-precipitation with affinity purified, subunit-specific antibodies to characterize and isolate 26S ad 26Si proteasomes; activity assays with model substrate peptides to characterize the catalytic properties of 26S and 26i active sites; and electron microscopy to visualize individual 20S and 20Si particles with the associated activators thereby allowing their classification and quantitation. In Aim 2, we will immuno-purify the 26S or 26Si proteasomes in a manner that traps their interaction with substrates, generate product peptides, identify them by mass spectrometry and perform bioinformatic analysis. We will establish the cell extraction procedure that most efficiently traps substrates at the 26S or 26i proteasomes; immuno-purify the proteasome/substrate complexes with subunit-specific antibodies; perform in vitro degradation of the co-purified substrates under conditions of single recruitment that most closely recapitulates degradation in vivo; isolate product peptides in a two step procedure that selects for peptides with normal and antigenic properties; and separate the peptide mixtures from any full length proteins that may dissociate from the proteasome. The peptide mixtures will be identified by mass spectrometry. PUBLIC HEALTH RELEVANCE: This fellowship will revitalize the applicant's research career by stimulating a major change in the direction of her research, broadening her scientific background, and acquiring new skills and capabilities in the area of Virology. She will establish an approach for the identification of large pools of natural peptides produced by 26S and the immune 26Si proteasome, and provide a proof of principle that this approach can identify viral subversion of specific cellular pathways and/or natural precursors of antigenic peptides. A successful proof of principle may allow this approach to be employed in other disease models and have a major scientific impact.

描述（由申请人提供）：该奖学金将通过刺激其研究方向的重大变化，扩大她的科学背景，并在病毒学领域获得新技能和能力来振兴申请人的研究职业。我们过去的工作集中在泛素 - 蛋白酶体系统调节非感染的单细胞生物体酿酒酵母的细胞生长和分裂的机制上。特别感兴趣的是，发现可以将底物捕获在26S蛋白酶体上，以使其后来的蛋白水解在受控的体外条件下，从而导致可以通过质谱法分析的肽的积累。我们未发表的数据表明，该策略可用于监测由哺乳动物26S蛋白酶体产生的大量天然产物肽及其免疫26SI版本，该版本由产生抗原肽的干扰素（IFN）刺激。当应用于病毒感染的细胞时，该分析可能揭示了病毒介导的蛋白酶体蛋白水解变化的特定“肽特征”，包括特定细胞途径的病毒颠覆和抗原肽的天然前体。这些主题在病毒学方面具有重要意义，但是通过传统方法解决这些主题的成功率很低。 我们将表征与从未感染的原代小鼠巨噬细胞和被痘病毒Ectromelia（EV）感染的巨噬细胞共纯化的天然底物产生的肽池的变化。 EV克服了巨噬细胞的IFN介导的抗病毒防御能力，这是其致病性的关键，可能取决于尚未确定的蛋白酶体形式。在AIM 1中，我们将在存在或不存在IFN的情况下以未感染和EV感染的巨噬细胞表达的26S和26SI蛋白酶体。我们将使用QPCR，定量的蛋白质印迹和免疫 - 周期化学来检测26S和26SI特定亚基的表达；具有亲和力纯化的，亚基特异性抗体的免疫原子，以表征和分离26S AD 26SI蛋白酶体；具有模型底物肽的活性测定，以表征26s和26i活性位点的催化特性；和电子显微镜可视化单个20S和20SI颗粒的颗粒，从而可以分类和定量。在AIM 2中，我们将以困扰其与底物相互作用，生成产品肽，通过质谱识别并执行生物信息学分析的方式来免疫纯化26S或26SI蛋白酶体。我们将建立最有效地捕获26S或26i蛋白酶体的底物的细胞提取程序；免疫纯化具有亚基特异性抗体的蛋白酶体/底物复合物；在最紧密地概括体内降解的单一募集条件下，在体外进行了体外降解；在两个步骤的过程中分离产物肽，该肽选择具有正常和抗原特性的肽；并将肽混合物与可能与蛋白酶体分离的任何全长蛋白分开。肽混合物将通过质谱法鉴定。 公共卫生相关性：该奖学金将通过刺激她的研究方向的重大变化，扩大她的科学背景并获得病毒学领域的新技能和能力来振兴申请人的研究生涯。她将建立一种方法，以鉴定由26S和免疫26SI蛋白酶体产生的大量天然肽，并提供了一种原理证明，证明这种方法可以识别抗原肽的特定细胞途径和/或天然前体的病毒颠覆。成功的原则证明可以允许这种方法用于其他疾病模型，并产生重大的科学影响。