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.
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