Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells

Benjamin E. Turk, Eric C. Griffith, Susan Wolf, Klaus Biemann, Yie Hwa Chang, Jun Liu

Research output: Contribution to journalArticle

Abstract

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 [35S]-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.

Original languageEnglish (US)
Pages (from-to)823-833
Number of pages11
JournalChemistry and Biology
Volume6
Issue number11
DOIs
StatePublished - Nov 1999
Externally publishedYes

Fingerprint

Endothelial cells
Methionine
Endothelial Cells
Processing
Pharmaceutical Preparations
Cell growth
Glyceraldehyde-3-Phosphate Dehydrogenases
Proteins
Cells
Growth
Cyclophilin A
Angiogenesis Inhibitors
methionine aminopeptidase 2
ovalicin
O-(chloroacetylcarbamoyl)fumagillol
Cell proliferation
Cell Extracts
Purification
Cell Proliferation
Cell Line

Keywords

  • Angiogenesis
  • Methionine aminopeptidase
  • Ovalicin
  • TNP-470

ASJC Scopus subject areas

  • Organic Chemistry

Cite this

Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells. / Turk, Benjamin E.; Griffith, Eric C.; Wolf, Susan; Biemann, Klaus; Chang, Yie Hwa; Liu, Jun.

In: Chemistry and Biology, Vol. 6, No. 11, 11.1999, p. 823-833.

Research output: Contribution to journalArticle

Turk, Benjamin E. ; Griffith, Eric C. ; Wolf, Susan ; Biemann, Klaus ; Chang, Yie Hwa ; Liu, Jun. / Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells. In: Chemistry and Biology. 1999 ; Vol. 6, No. 11. pp. 823-833.
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AU - Turk, Benjamin E.

AU - Griffith, Eric C.

AU - Wolf, Susan

AU - Biemann, Klaus

AU - Chang, Yie Hwa

AU - Liu, Jun

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N2 - 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 [35S]-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.

AB - 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 [35S]-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.

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