Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells.
BACKGROUND
The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however.


RESULTS
Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation.


CONCLUSIONS
Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.
Turk
B E
BE
Department of Chemistry, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Griffith
E C
EC
Wolf
S
S
Biemann
K
K
Chang
Y H
YH
Liu
J O
JO
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D001011:Aorta; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003510:Cyclohexanes; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism; D000077609:O-(Chloroacetylcarbamoyl)fumagillol; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012717:Sesquiterpenes / Q000494:pharmacology*; D014471:Umbilical Veins
D020533:Angiogenesis Inhibitors; D003510:Cyclohexanes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D019814:Myristic Acid; C002781:ovalicin; D008715:Methionine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000626:Aminopeptidases; C106541:methionine aminopeptidase 2; D008666:Metalloendopeptidases; D000077609:O-(Chloroacetylcarbamoyl)fumagillol