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Endosomal proteolysis of insulin‐like growth factor‐I at its C‐terminal D‐domain by cathepsin B
Author(s) -
Authier François,
Kouach Mostafa,
Briand Gilbert
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.06.066
Subject(s) - endosome , cathepsin o , cathepsin b , proteolysis , cathepsin d , biochemistry , cathepsin l1 , protease , cathepsin h , cathepsin , chemistry , cathepsin s , microbiology and biotechnology , enzyme , biology , intracellular
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 (IC 50 ≈ 1.5 μM). 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 Lys 68 ‐Ser 69 , Ala 67 ‐Lys 68 , Pro 66 ‐Ala 67 and Lys 65 ‐Pro 66 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 [ 125 I]IGF‐I and, in vitro, the degradation of exogenous [ 125 I]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.