IDENTIFICATION OF IN VIVO SUBSTRATES OF SERINE PROTEASE QPP BY MASS SPECTROMETR

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。静止细胞脯氨酸二肽酶(QPP)是一种从蛋白质的氨基末端裂解二肽的丝氨酸蛋白酶。在静止期(G0)细胞中抑制QPP活性或表达会导致细胞周期进展和诱导细胞凋亡。推测QPP参与维持G0细胞的存活。基于对合成底物的研究，QPP从蛋白质的氨基末端切割双肽，其中Pro是倒数第二个氨基酸残基。然而，QPP的体内蛋白底物尚不清楚。这项工作的重点是通过鉴定该酶的蛋白质底物来促进对QPP活性的理解。为此，野生型和酶促死亡的QPP变异体被稳定地导入细胞系。将含有6X His c-末端标签的潜在底物的构建载体导入到这些细胞中，用于在固定的金属离子柱上进行亲和纯化。转染后，收集细胞上清液，在金属离子亲和层析柱上浓缩蛋白，并用咪唑缓冲液洗脱。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离蛋白质，从凝胶中切下蛋白质带。切下的蛋白质用蛋白酶在凝胶中消化，释放出N-末端的多肽。然后用MALDI-TOF质谱仪分析这些多肽，以确定野生型QPP表达细胞中是否发生了氨基末端加工。我们首先研究了两种潜在的底物：神经肽y(NPY)和白介素2(IL-2)，这两种物质都含有潜在的QPP加工位点。在SDS-PAGE中，我们鉴定了一种与IL-2接近的协同纯化蛋白--血红蛋白。血红蛋白来源于培养细胞所用的血清。使用无血清培养液不支持细胞的生长，因此我们目前正专注于在SDS-PAGE上更好地分离紧密排列的IL-2和血红蛋白，以便可以单独切除IL-2带。还在努力提高NPY样品的产量，以便通过质谱学进行检测。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Quiescent cell prolyl di-peptidase (QPP) is a serine protease that cleaves di-peptides from the amino terminus of proteins. Inhibition of QPP activity or expression in quiescent (G0) cells leads to cell cycle progression and apoptosis induction. It is hypothesized that QPP is involved in maintaining the survival of G0 cells. Based on work with synthetic substrates, QPP cleaves di-peptides from the amino terminus of proteins where proline is the penultimate amino acid residue. However, the in vivo protein substrates of QPP are unknown. This work is focused on advancing the understanding of QPP activity by identification of protein subtrates of the enzyme. To this end, wild-type and enzymatically dead variants of QPP are stably transfected into cell lines. To these cells are transfected constructs of potential substrates which have 6X His c-terminal tags for affinity purification on immobilized metal ion columns. Following transfection, cell supernates are collected and the proteins are enriched on the metal ion affinity columns and eluted with imidazole buffer. The proteins are separated by SDS-PAGE and protein bands are excised from the gels. Excised proteins are digested in-gel with proteases to liberate the n-terminal peptide. The peptides are then analyzed by MALDI-TOF mass spectrometry to determine if amino-terminal processing has occurred in the wild-type QPP expressing cells. We have begun this analysis by looking at two potential substrates: neuropeptide y (NPY) and interleukin 2 (IL-2), both of which contain a potential QPP processing site. We have identified a co-purifying protein, hemoglobin, which migrates close to IL-2 in SDS-PAGE. The hemoglobin originates from the serum used to culture the cells. Use of serum-free media does not support the growth of the cells, so we are currently focusing on better separating the closely-spaced IL-2 and hemoglobin on SDS-PAGE so the IL-2 band can be excised separately. Efforts are also being made to increase the yield of the NPY samples to allow detection by mass spectrometry.