Determination of intracellular prolyl/glycyl proteases in intact living human cells and protoporphyrin IX production as a reporter system.
The determination of enzyme activity or inhibition in intact living cells is a problem in the development of inhibitors for intracellular proteases. The production of fluorescent protoporphyrin IX (PpIX) from the nonfluorescent (N)-Gly/Pro-5-aminolevulinic acid (ALA) substrates was used to evaluate the prolyl/glycyl-specific dipeptidylpeptidase IV (DPPIV)-like and prolyloligopeptidase (POP)-like activities of human cells. The results demonstrated that whereas POP-like activity could be attributed to the actual POP, the DPPIV-like activity could be related to actual DPPIV only in one colon cell line. In the other breast and colon cell lines, DPPIV-like activity was intracellular and displayed by other prolyl-specific aminopeptidases. Our experiments also demonstrated the involvement of glycyl-specific proteases in the processing of ALA precursors. These observations have important consequences for the development and evaluation of selective inhibitors for these enzymes.
Berger
Yann
Y
University Institute of Pathology, CHUV, Bugnon 25, 1011 Lausanne, Switzerland.
Chapuis Bernasconi
Catherine
C
Schmitt
Frédéric
F
Neier
Reinhard
R
Juillerat-Jeanneret
Lucienne
L
D000243:Adenosine Deaminase / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000201:enzymology / Q000473:pathology; D003546:Cysteine Endopeptidases / Q000032:analysis*; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D015195:Drug Design; D005260:Female; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D008722:Methods; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D011524:Protoporphyrins / Q000032:analysis / Q000096:biosynthesis*; D012697:Serine Endopeptidases / Q000032:analysis*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D006023:Glycoproteins; D011480:Protease Inhibitors; D011524:Protoporphyrins; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX; C042807:DPP4 protein, human; D018819:Dipeptidyl Peptidase 4; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases; D003546:Cysteine Endopeptidases; C050874:glycyl endopeptidase; D000243:Adenosine Deaminase