Determination of intracellular prolyl/glycyl proteases in intact living human cells and protoporphyrin IX production as a reporter system.

SummaryThe determination of enzyme activity or inhibition inintact living cells is a problem in the development ofinhibitors for intracellular proteases. The productionof fluorescent protoporphyrin IX (PpIX) from the non-fluorescent (N)-Gly/Pro-5-aminolevulinic acid (ALA)substrates was used to evaluate the prolyl/glycyl-spe-cific dipeptidylpeptidase IV (DPPIV)-like and prolyloli-gopeptidase (POP)-like activities of human cells. Theresults demonstrated that whereas POP-like activitycould be attributed to the actual POP, the DPPIV-likeactivity could be related to actual DPPIV only in onecolon cell line. In the other breast and colon cell lines,DPPIV-like activity was intracellular and displayed byother prolyl-specific aminopeptidases. Our experimentsalso demonstrated the involvement of gylcyl-specificproteases in the processing of ALA precursors.These observations have important consequencesfor the development and evaluation of selective inhib-itors for these enzymes.Introduction Proteases are major targets in the treatment of humandiseases. However, although there has been much suc-cess with drugs developed for inhibiting extracellularproteases (either secreted or inserted in the outer cellmembrane), the inhibition of intracellular proteases,and the evaluation of this inhibition, in intact living cellsis difficult to achieve due to the limitation of membranepermeability of protease substrates or inhibitors. It isbecoming increasingly accepted that intracellular pro-teases are important targets for drug development;thus, it is necessary to develop tools for evaluating theeffects of inhibitors in the intracellular environment ofthese enzymes. Most present approaches, however, in-volve cell lysis, thus losing important information on thestate of living cells.Many biologically active peptides are protected fromgeneral proteolytic degradation by evolutionarily con-served Pro. This protection is provided due to confor-