Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B

FEBS Lett. 2005 Aug 15;579(20):4309-16. doi: 10.1016/j.febslet.2005.06.066.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • Cathepsin B / antagonists & inhibitors
  • Cathepsin B / metabolism*
  • Cysteine Proteinase Inhibitors / pharmacology
  • Dipeptides / pharmacology
  • Endosomes / enzymology*
  • Humans
  • Insulin-Like Growth Factor I / chemistry
  • Insulin-Like Growth Factor I / metabolism*
  • Leucine / analogs & derivatives
  • Leucine / pharmacology
  • Protein Structure, Tertiary
  • Tumor Cells, Cultured

Substances

  • Cysteine Proteinase Inhibitors
  • Dipeptides
  • N-(3-propylcarbamoyloxirane-2-carbonyl)-isoleucyl-proline
  • Insulin-Like Growth Factor I
  • Cathepsin B
  • Leucine
  • E 64