Poxvirus-mediated change to proteasomal peptide signatures in macrophages

描述(由申请人提供)：该奖学金将通过促进申请人研究方向的重大变化，拓宽她的科学背景，并获得病毒学领域的新技能和能力，来重振申请人的研究生涯。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生物酿酒酵母的细胞生长和分裂的机制上。特别令人感兴趣的是，发现底物可以被捕获在26S蛋白酶体上，从而允许它们稍后在体外条件下进行蛋白质分解，从而导致多肽的积累，可以用质谱仪进行分析。我们未发表的数据表明，这一策略可以用于监测哺乳动物26S蛋白酶体及其免疫26Si版本产生的大量天然产物多肽库，这些多肽是由产生抗原肽的干扰素(IFN)刺激的。当应用于病毒感染的细胞时，这一分析可能揭示病毒介导的蛋白酶体蛋白分解变化的特殊“肽特征”，包括病毒对特定细胞途径和抗原肽的天然前体的颠覆。这些主题在病毒学中具有重大意义，但通过传统方法解决这些问题的成功率很低。我们将研究从未感染的原代小鼠巨噬细胞和感染了痘病毒皮疹(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多肽池的变化。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，可能依赖于一种尚未确定的蛋白酶体形式。在目标1中，我们将研究在存在或不存在IFN的情况下，未感染和EV感染的巨噬细胞中表达的26S和26Si蛋白酶体的特征。我们将使用qPCR、定量Western印迹和免疫细胞化学来检测26S和26Si特异性亚基的表达；利用亲和纯化的亚基特异性抗体进行免疫沉淀来鉴定和分离26S ad 26Si蛋白酶体；使用模型底物多肽进行活性分析来表征26S和26I活性位点的催化特性；以及电子显微镜来显示单个20S和20Si颗粒与相关激活剂的关系，从而使它们能够分类和定量。在目标2中，我们将以一种捕获它们与底物相互作用的方式对26S或26Si蛋白酶体进行免疫纯化，产生产物多肽，通过质谱仪对它们进行鉴定，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26S或26I蛋白酶体上的细胞提取程序；免疫纯化具有亚基特异性抗体的蛋白酶体/底物复合体；在最接近体内降解的单次招募条件下进行共纯化底物的体外降解；通过两步程序分离产品多肽，选择具有正常和抗原性的多肽；并将多肽混合物与任何可能从蛋白酶体分离的全长蛋白质分离。多肽混合物将通过质谱学进行鉴定。

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 Our unpublished data suggest that this strategy could be used to monitor large pools of natural product peptides generated by mammalian 26S proteasome and its immune 26Si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