Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B.
IGF-I is degraded within the endosomal apparatus as a consequence of receptor-mediated endocytosis. However, the nature of the responsible protease and the position of the cleavage sites in the IGF-I molecule remain undefined. In vitro proteolysis of IGF-I using an endosomal lysate required an acidic pH and was sensitive to CA074, an inhibitor of the cathepsin B enzyme. By nondenaturing immunoprecipitation, the acidic IGF-I-degrading activity was attributed to the luminal species of endosomal cathepsin B with apparent molecular masses of 32- and 28-kDa. The cathepsin B precursor, procathepsin B, was processed in vitro within isolated endosomes at pH 5 or at 7 in the presence of ATP, the substrate of the vacuolar H(+)-ATPase. The rate of IGF-I hydrolysis using an endosomal lysate or pure cathepsin B was found to be optimal at pH 5-6 and moderate at pH 4 and 7. Competition studies revealed that EGF and IGF-I share a common binding site on the cathepsin B enzyme, with native IGF-I displaying the lowest affinity for the protease (IC50 approximately 1.5 microM). Hydrolysates of IGF-I generated at low pH by endosomal IGF-I-degrading activity and analyzed by reverse-phase HPLC and mass spectrometry revealed cleavage sites at Lys68-Ser69, Ala67-Lys68, Pro66-Ala67 and Lys65-Pro66 within the C-terminal D-domain of IGF-I. Treatment of human HepG2 hepatoma cells with the cathepsin B proinhibitor CA074-Me reduced, in vivo, the intracellular degradation of internalized [125I]IGF-I and, in vitro, the degradation of exogenous [125I]IGF-I incubated with the cell-lysates at pH 5. Inhibitors of cathepsin B and pro-cathepsin B processing, which abolish endosomal proteolysis of IGF-I and alter tumor cell growth and IGF-I receptor signalling, merit investigation as antimetastatic drugs.
Authier
François
F
Institut National de la Santé et de la Recherche Médicale U510, Faculté de Pharmacie, Paris XI, 5 Rue Jean Baptiste Clement, 92296 Châtenay-Malabry, France. francois.authier@cep.u-psud.fr
Kouach
Mostafa
M
Briand
Gilbert
G
D002384:Catalysis; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D011992:Endosomes / Q000201:enzymology*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D014407:Tumor Cells, Cultured
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; C068250:N-(3-propylcarbamoyloxirane-2-carbonyl)-isoleucyl-proline; D007334:Insulin-Like Growth Factor I; D002401:Cathepsin B; D007930:Leucine; C024974:E 64