QPP: Protease that Prevents Apoptosis in Quiescent Cells

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EXCEED THE SPACE PROVIDED. QPP (Quiescent cell Proline di-Peptidase) (DPP7) is a protease hypothesized to control the survival of lymphocytes and neuronal cells that are in the GO stage of cell cycle, because QPP inhibitors induce apoptosis in these cells. I. The construction of QPP mutant mice provides a direct approach for defining the functional significance of QPP in vivo. Four systems will be used which will allow a broad analysis of the role of QPP in various cell types and stages of development: (a) Conventional QPP-/- mice will be analyzed for defects in development. (b) If the QPP-/- genotype is embryonically lethal, lymphoid development will be tested by fetal liver transfer into RAG-2-/- mice. (c) If lethality of the QPP-/- genotype occurs before lymphoid stern cell development, chimeric mice will be generated, using the RAG-2-deficient blastocyst complementation system that allows expression of the homozygous QPP mutation in the lymphoid lineage only. (d) To define the role of QPP in mature cells and individual organs, conditional QPP ko mice will be engineered, using the Cre-loxP recombination system that provides control of expression of the homozygous mutation. I1. Inhibition of QPP expression in vitro will be carried out to define the role of QPP in human lymphocytes and neuronal cells. Four systems will be employed: (a) Since the QPP protease activity requires dimerization, enzyme active site mutants will be screened for dominant negative activity. (b) RNA interference (RNAi) will be used for silencing QPP transcripts. (c) Antisense oligos will be tested for blocking QPP protein expression in primary lymphocytes and neuronal cells. (d) A full-length antisense QPP cDNA in a recombinant adenovirus will be introduced into human and mouse cell lines and primary cells to block expression of QPPo II1. Characterization of QPP substrate(s) will be carried out to understand the mechanism and/or pathway of QPP-rnediated survival of GO cells. (a) Based on the working hypotheses, LKLF and neurotrophins, as well as novel candidates, will be screened as potential substrates of QPP. (b) To confirm that cleavage of a candidate substrate by QPP in vitro is functionally significant, co-localization experiments will be carried out. Collectively, these studies will lead to a better understanding of the constitutive GO survival program in eukaryotic cells in vivo and in vitro. In addition, they will provide a tool for the analysis of the default PCD pathway in quiescent cells. PERFORMANCE SITE ========================================Section End===========================================