Poxvirus-mediated change to proteasomal peptide signatures in macrophages

描述（由申请人提供）：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，振兴申请人的研究生涯。我们过去的工作主要集中在泛素-蛋白酶体系统调节非感染性单细胞酿酒酵母细胞生长和分裂的机制。特别令人感兴趣的是发现底物可以以允许其在受控的体外条件下随后蛋白水解的方式被捕获在26 S蛋白酶体处，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，这种策略可用于监测由哺乳动物26 S蛋白酶体及其免疫26 Si版本产生的天然产物肽的大池，所述26 Si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，这种分析可能会揭示一个特定的“肽签名”的病毒介导的蛋白酶体蛋白水解的变化，包括病毒颠覆特定的细胞途径和抗原肽的天然前体。这些主题在病毒学中具有重要意义，但通过传统方法解决它们的成功率很低。 我们将表征从天然底物与26 S或26 Si蛋白酶体共纯化的肽池中的变化，所述蛋白酶体来自未感染的原代小鼠巨噬细胞和感染痘病毒鼠痘（EV）的巨噬细胞。EV克服了IFN介导的巨噬细胞的抗病毒防御，这是其致病性的关键，并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IFN存在或不存在下在未感染和EV感染的巨噬细胞中表达的26 S和26 Si蛋白酶体。我们将使用qPCR、定量western blot和免疫细胞化学检测26 S和26 Si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26 S和26 Si蛋白酶体;使用模型底物肽进行活性测定以表征26 S和26 i活性位点的催化性质;和电子显微镜观察具有相关活化剂的单个20 S和20 Si颗粒，从而允许它们的分类和定量。在目标2中，我们将以捕获它们与底物相互作用的方式免疫纯化26 S或26 Si蛋白酶体，产生产物肽，通过质谱法鉴定它们并进行生物信息学分析。我们将建立最有效地将底物捕获在26 S或26 i蛋白酶体上的细胞提取程序;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一募集条件下进行共纯化底物的体外降解;在两步程序中分离产物肽，所述两步程序选择具有正常和抗原性质的肽;以及将肽混合物与可能从蛋白酶体解离的任何全长蛋白质分离。将通过质谱法鉴别肽混合物。

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 Our unpublished data suggest that this strategy could be used to monitor large pools of natural product peptides generated by mammalian 26S proteasome and its immune 26Si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.