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 Our unpublished data suggest that this strategy could be used to monitor large pools of natural product peptides generated by mammalian 26S proteasome and its immune 26Si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申请人研究方向的重大转变、拓宽她的科学背景以及获得病毒学领域的新技能和能力，从而重振申请人的研究生涯。我们过去的工作重点是泛素蛋白酶体系统调节非感染性单细胞生物酿酒酵母的细胞生长和分裂的机制。特别令人感兴趣的是，底物可以被捕获在 26S 蛋白酶体上，其方式允许它们随后在受控的体外条件下进行蛋白水解，从而导致可以通过质谱分析的肽的积累。我们未发表的数据表明，该策略可用于监测由产生抗原肽的干扰素 (IFN) 刺激的哺乳动物 26S 蛋白酶体及其免疫 26Si 版本产生的大量天然产物肽。当应用于病毒感染的细胞时，该分析可能揭示病毒介导的蛋白酶体蛋白水解变化的特定“肽特征”，包括病毒对特定细胞途径和抗原肽天然前体的颠覆。这些主题在病毒学中具有重要意义，但通过传统方法解决这些问题的成功率很低。 我们将表征从未感染的原代小鼠巨噬细胞和感染痘病毒 ectromelia (EV) 的巨噬细胞中与 26S 或 26Si 蛋白酶体共纯化的天然底物产生的肽库的变化。 EV克服了巨噬细胞的干扰素介导的抗病毒防御，这是其致病性的关键，并且可能依赖于一种尚未确定的蛋白酶体形式。在目标 1 中，我们将表征在存在或不存在 IFN 的情况下未感染和 EV 感染的巨噬细胞中表达的 26S 和 26Si 蛋白酶体。我们将使用qPCR、定量蛋白质印迹和免疫细胞化学来检测26S和26Si特异性亚基的表达；使用亲和纯化的亚基特异性抗体进行免疫沉淀，以表征和分离 26S 和 26Si 蛋白酶体；使用模型底物肽进行活性测定，以表征 26S 和 26i 活性位点的催化特性；和电子显微镜观察单个 20S 和 20Si 颗粒以及相关的活化剂，从而对其进行分类和定量。在目标 2 中，我们将以捕获 26S 或 26Si 蛋白酶体与底物相互作用的方式免疫纯化它们，生成产物肽，通过质谱法识别它们并进行生物信息学分析。我们将建立最有效地捕获 26S 或 26i 蛋白酶体底物的细胞提取程序；使用亚基特异性抗体免疫纯化蛋白酶体/底物复合物；在最接近体内降解的单次补充条件下进行共纯化底物的体外降解；通过两步程序分离产物肽，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解离的任何全长蛋白质分开。肽混合物将通过质谱法进行鉴定。 公共卫生相关性：该奖学金将通过刺激申请人研究方向的重大转变、拓宽她的科学背景以及获得病毒学领域的新技能和能力，从而重振申请人的研究生涯。她将建立一种方法来鉴定 26S 和免疫 26Si 蛋白酶体产生的大量天然肽，并提供原理证明，证明该方法可以识别特定细胞途径和/或抗原肽天然前体的病毒颠覆。成功的原理证明可能会让这种方法应用于其他疾病模型，并产生重大的科学影响。