Targeted gene disruption of methionine aminopeptidase 2 results in an embryonic gastrulation defect and endothelial cell growth arrest

Abstract

The antiangiogenic agent fumagillin (Fg) and its analog TNP-470 bind to intracellular metalloprotease methionine aminopeptidase-2 (MetAP-2) and inhibit endothelial cell growth in a p53-dependent manner. To confirm the role of MetAP-2 in endothelial cell proliferation and to validate it as a physiological target for the Fg class of antiangiogenic agents, we have generated a conditional MetAP-2 knockout mouse. Ubiquitous deletion of the MetAP-2 gene (MAP2) resulted in an early gastrulation defect, which is bypassed in double MetAP-2͞p53 knockout embryos. Targeted deletion of MAP2 specifically in the hemangioblast lineage resulted in abnormal vascular development, and these embryos die at the midsomite stage. In addition, knockdown of MetAP-2 using small interfering RNA or homologous recombination specifically suppresses the proliferation of cultured endothelial cells. Together, these results demonstrate an essential role for MetAP-2 in angiogenesis and indicate that MetAP-2 is responsible for the endothelial cell growth arrest induced by Fg and its derivatives.angiogenesis ͉ TNP-470 T he methionine aminopeptidase (MetAPs) family of cytosolic metalloproteases are responsible for the cleavage of the initial methionine from the N termini of nascent proteins (1, 2). Eukaryotes express two forms of MetAPs, types 1 and 2, which possess similar in vitro substrate specificities. Removal of the N-terminal methionine by MetAP activity is important for subsequent Nterminal modifications, such as myristoylation (3) and acetylation (4). In addition, MetAP activity may affect protein stability according to the N-end rule proposed by Varshavsky and coworkers (5). It has been shown that MetAP function is essential for growth and survival of prokaryotes (which express only MetAP-1) and of the budding yeast Saccharomyces cerevisiae (6, 7). However, the physiological roles of MetAPs have not yet been identified in vertebrates.MetAP-2 is specifically inhibited by TNP-470, a synthetic derivative of the Aspergillus fumigatus natural product fumagillin (Fg; see refs. 8 and 9). TNP-470 selectively inhibits endothelial cell growth in vitro with picomolar efficacy and has a potent antiangiogenic effect in vivo (10, 11). Because of the requirement of angiogenesis for solid tumor growth, MetAP-2 is currently being targeted with small-molecule inhibitors for anticancer and other angiogenesisrelated diseases therapy, even though the mode of action of TNP-470 is not entirely clear (12-16). Several lines of evidence suggest that MetAP-2 is important for the antiangiogenic effect of TNP-470. First, we have recently demonstrated that an A362T variant of human MetAP-2 confers resistance to the cytostatic effect of a Fg analog in MAP1-null yeast (17). In addition, it has been shown that the ability of various Fg analogues to inhibit endothelial cell growth correlates with their MetAP-2 enzymatic inhibitory activity in vitro (9). However, a recent report using RNA interference (RNAi)-mediated MetAP-2 down-regulation contradicts our results and thus r...

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

“…If it is not the case, this further supports the fact that MetAP2, the enzyme in charge of this cleavage, is more limiting in humans than in plants (71). This could explain why it is possible to inactivate this enzyme in plants and not in animals (72,73). Of note, these polypeptides retaining the first Met, although they do have a Thr or a Val at position 2, were found to be NAAed on the Met residue that does not correspond to a clear Nat target (14).…”

Correlation of tumor growth suppression and methionine aminopetidase-2 activity blockade using an orally active inhibitor

“…Antisense and siRNA also have been used to examine the role of MetAP2. Knockdown of MetAP2 results in cell growth inhibition and apoptosis in endothelial and tumor cells (13)(14)(15). The antiproliferative phenotype of MetAP2 knockdown can be recapitulated by the inhibition of the enzymatic activity of MetAP2.…”

“…That MetAP2 is a bifunctional protein complicates the study of its function with deletion or siRNA techniques, because the eIF-2␣ has an important role in controlling protein synthesis and cell growth (8). Recently, it has been reported that MetAP2 knockout mouse embryos fail to undergo gastrulation, and targeted knockout of MetAP2 in the hemangioblast lineage caused abnormal vascular development and a lethal phenotype at the midsomite stage (13). These results clearly demonstrate an essential role of MetAP2 in embryonic development and vasculogenesis.…”

Ascending dose‐controlled trial of beloranib, a novel obesity treatment for safety, tolerability, and weight loss in obese women

“…Inhibitors of methionine aminopetidase 2 (MetAP2), originally developed as anti-angiogenic agents for the treatment of cancer (9), are a novel mechanism for inducing significant and sustained weight loss in animal models of obesity at low doses that do not impact angiogenesis (10)(11)(12)(13). Fumagillin, a natural product isolated from aspergillus fumigatus, is a potent and selective MetAP2 inhibitor (14).…”