Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction.
Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1-10 μM). The protein kinase A inhibitor KT5720 (1 μM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction.
Department of Cell Biology and Development, Biomedical Sciences Institute, University of São Paulo, São Paulo, Brazil.
Lilian C
LC
Russo
Leandro M
LM
Castro
Fabio C
FC
Gozzo
Emer S
ES
Ferro
D000595:Amino Acid Sequence; D002227:Carbazoles / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D002227:Carbazoles; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; C057416:KT 5720; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D007545:Isoproterenol