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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。静止细胞脯氨酰二肽酶（QPP）是一种丝氨酸蛋白酶，从蛋白质的氨基末端切割二肽。抑制静止期（G 0）细胞中的QPP活性或表达导致细胞周期进展和凋亡诱导。假设QPP参与维持G 0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二肽，其中脯氨酸是倒数第二个氨基酸残基。然而，QPP的体内蛋白质底物是未知的。这项工作的重点是通过鉴定酶的蛋白质亚基来推进对QPP活性的理解。为此，将QPP的野生型和酶促死亡变体稳定转染到细胞系中。向这些细胞中转染潜在底物的构建体，所述潜在底物具有用于在固定化金属离子柱上亲和纯化的6X His C-末端标签。转染后，收集细胞上清液，并在金属离子亲和柱上富集蛋白质，并用咪唑缓冲液洗脱。通过SDS-PAGE分离蛋白质，并从凝胶上切下蛋白质条带。用蛋白酶在凝胶中消化切除的蛋白质以释放N-末端肽。然后通过MALDI-TOF质谱法分析肽以确定在表达野生型QPP的细胞中是否发生氨基末端加工。我们已经开始了这项分析，通过寻找两个潜在的底物：神经肽y（NPY）和白细胞介素2（IL-2），这两个都含有一个潜在的QPP加工位点。我们已经鉴定了共纯化蛋白质，血红蛋白，其在SDS-PAGE中迁移接近IL-2。血红蛋白来源于用于培养细胞的血清。使用无血清培养基不支持细胞的生长，因此我们目前专注于在SDS-PAGE上更好地分离紧密间隔的IL-2和血红蛋白，以便可以单独切除IL-2条带。也正在努力提高NPY样品的产量，以允许通过质谱法进行检测。

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.