The post-translational processing of initiator methionine during protein synthesis is a universal biological process that is conserved from prokaryotes to eukaryotes. Methionine aminopeptidases are the enzymes catalyzing the removal of initiator methionine. One gene is known in prokaryotes while two genes are known in eukaryotes encoding methionine aminopeptidases. The importance of initiator methionine processing is underscored by the fact that deletion of the methionine aminopeptidase genes in either prokaryotes or eukaryotes is lethal. Of the two methionine aminopeptidase genes in eukaryotes, the type 2 enzyme (MetAP2) has been shown to be the direct target for the fumagillin family of angiogenesis inhibitors, including its analog TNP-470. Work in the past several years has provided strong evidence that MetAP2 is a physiologically relevant target for TNP-470. It has also been found that inhibition of endothelial cell proliferation by TNP-470 is mediated by the tumor suppressor gene p53. Thus, TNP-470 is capable of activating p53, which induces the expression of p21that is responsible for the cell cycle blockade of endothelial cells. These studies reveal a unique role of MetAP2 in the progression of the endothelial cell cycle. Recently, a novel anticancer drug entering Phase II clinical trial known as bengamide was found to inhibit both MetAP2 and MetAP1 in vitro and to block cell cycle in both G1 and G2/M phase. It is hypothesized that MetAP1 may play a role in the cell cycle at the G2/M phase. The major objective of the current proposal is to further delineate the physiological functions of MetAP1 and MetAP2 employing yeast as a model system and to identify isoform-specific inhibitors for MetAP1 and MetAP2 by high throughput screens. The functions of the different domains in MetAP1 and MetAP2 will be investigated by creating various yeast mutants expressing different domains of these enzymes and determining the phenotypic changes using DNA microarray. The different known activators of p53 will be systematically examined to identify potential mediators of p53 activation by TNP-470. Molecular probes of bengamide will be prepared to confirm its interaction with MetAP1 and MetAP2. High throughput screens will be conducted to identify specific inhibitors for MetAP1 and MetAP2. The newly identified inhibitors for MetAP1 will be employed to assess the physiological role of MetAP1 in the cell cycle progression in the G2/M phase.
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