Focused proteomic analysis of functional network for peptidyl prolyl cis/trans isomerases

肽基脯氨酰顺/反式异构酶(PPlase)催化绕着Pro之前的肽键旋转，这一步骤对新合成的蛋白质的折叠可能是限速的。PPlase还能够结合许多蛋白质，并充当伴侣；因此，它们被认为调节细胞内蛋白质的折叠、组装和运输，并控制蛋白质的活动。免疫抑制药物环孢素A(CsA)和FK506分别与亲环素(CYP)和FK506结合蛋白(FKBP)结合，并抑制其同源PPlase活性，PPlase是最常见的靶标。CYP和FKBP都是普遍存在的，从细菌到人类都高度表达和保守。PPlase的第三个家族，小球蛋白，既不是CsA也不是FK506的靶标，但在细菌和人类之间也是保守的。尽管在几乎所有的生物体中都发现了一些CYP、FKBP和小球蛋白同源物，但大多数同源物的细胞功能仍有待挖掘。在这项研究中，我们利用蛋白质组学的方法系统地分析了23种不同的人类PPlase的潜在底物，包括9种CYP，11种FKBP和3种小球蛋白同源物，包括表位标记的PPlase的表达，有或没有PPlase抑制剂的亲和纯化，基于质谱学的蛋白质鉴定，以及数据库搜索。我们确定了这三种PPlase的大约200个潜在底物，并构建了PPlase的功能蛋白质网络。该网络包括在翻译、细胞周期、增殖、转录、应激反应、DNA/蛋白质代谢、RNA加工、蛋白质运输和核糖体生物发生中发挥作用的蛋白质。我们进一步研究了PPlase在核糖体生物发生中的作用，发现PPlase可能在人类核糖体生物发生的不同阶段发挥作用。

Peptidyl prolyl cis/trans isomerases (PPlases) catalyze the rotation about the peptide bond preceding proline, a step that can be rate-limiting for the folding of newly synthesized proteins. PPlases also have the ability to bind many proteins and to act as chaperones; thus they are believed to regulate folding, assembly and trafficking, and controlling activity of proteins in the cell. PPlases are most familiar as the targets of the immunosuppressive drugs, cyclosporin A (CsA) and FK506, which bind, respectively, to cyclophilin (CyP) and FK506-binding protein (FKBP) and inhibit their cognate PPlaseactivities. Both CyP and FKBP are ubiquitous, highly expressed and conserved from bacteria to human. The third family of PPlases, parvulins, is the target of neither CsA nor FK506, but is also conserved from bacteria to human. Although a number of CyP, FKBP and parvulin homologs have been identified in almost all organisms, the cellular functions of most of those homologs remain to be exploded. In this study, we analyzed systematically potential substrate for 23 different human PPlases including 9 CyP, 11 FKBP, and 3 parvulin homologs by using proteomic approaches that include expression of epitope-tagged PPlase, affinity purification in the presence or absence of PPlase inhibitor, mass-spectrometry based protein identification, and database searching. We identified about 200 potential substrates for the three types of PPlases and constructed a functional protein network for the PPlases. The network included proteins that have roles in translation, cell cycle, proliferation, transcription, stress-responses, DNA/protein metabolism, RNA processing, protein trafficking, and ribosome biogenesis. We investigated further the involvement of PPlases in ribosome biogenesis, and found that parvulin 14, FKBP25, and CyPB have probably roles in different stages of human ribosome biogenesis.