Steady-State Kinetic Characterization of Substrates and Metal-Ion Specificities of the Full-Length and N-Terminally Truncated Recombinant Human Methionine Aminopeptidases (Type 2)

Yang, Guang; Kirkpatrick, Robert B.; Ho, Thau; Zhang, Guifeng; Liang, Po‐Huang; Johanson, Kyung; Casper, David J.; Doyle, Michael L.; Marino, Joseph P.; Thompson, Scott K.; Chen, Wenfang; Tew, David G.; Meek, Thomas D.

doi:10.1021/bi010806r

Cited by 72 publications

(86 citation statements)

References 41 publications

(42 reference statements)

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“…MetAP2 is a protein with dual functions: MetAP activity and the stabilization of eIF-2a as p67 (Datta, 2000). Truncation of the highly charged N-terminal domain, speculated to be involved in the binding of eIF-2a and preventing its inactivation (Datta, 2000), does not affect MetAP2 enzyme activity in vitro (Yang et al, 2001;Wang et al, 2003b). Similarly, MetAP2 covalently inactivated by TNP-470 is still able to stabilize eIF-2a (Griffith et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation

et al. 2008

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Methionine aminopeptidase-2 (MetAP2) processes N-terminal methionine from nascent cellular proteins. Inhibition of MetAP2 has been shown to block angiogenesis and suppress tumor growth in preclinical tumor models. However, the biological role of MetAP2 in cancer is not well understood. We examined the effect of three distinct chemical classes of MetAP2 inhibitors on the growth of a panel of human cancer cells in vitro. All MetAP2 inhibitors caused inhibition of tumor cell growth in both anchorage-dependent and, particularly, in anchorage-independent manner. These data prompted us to examine the possible roles of MetAP2 in cancers. Ectopic expression of MetAP2 in NIH-3T3 cells caused transformation, evidenced by the formation of foci in monolayer culture and growth of large colonies in soft agar. Overexpression of MetAP2 in an immortalized bronchial epithelial cell line NL20 accelerated growth. These phenotypes induced by the overexpression of MetAP2 were reversed by the treatment with MetAP2 inhibitors, indicating that the catalytic function of MetAP2 was essential. Accordingly, overexpression of a catalytically inactive MetAP2 resulted in growth retardation of HT1080 tumor cells, suggesting a dominant-negative role of the inactive MetAP2 mutant. Finally, we analysed the expression of MetAP2 in patient cancer samples by immunohistochemistry. Moderate-to-high staining was identified in the majority of breast, colon, lung, ovarian and prostate carcinomas examined. These data suggest that MetAP2 plays an important role in tumor cell growth and may contribute to tumorigenesis.

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Section: Discussionmentioning

confidence: 99%

Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation

et al. 2008

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“…The fluorogenic substrate Met-AMC was purchased from Bachem. In the metal-titration experiments, the enzyme activities were monitored with a fluorescence assay by using Met-AMC (14,49) Data Collection and Structural Refinement. The personnel at the Protein Structure Laboratory at the University of Kansas assisted in collecting the reflection data.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of catalysis by monometalated methionine aminopeptidase

Xie

Qiang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.metalloprotease ͉ protein structure ͉ enzyme inhibition ͉ drug discovery ͉ metal occupancy A ll newly synthesized proteins have an amino-terminal methionine residue corresponding to the start codon AUG. In a significant number of cases, this initiating methionine residue is removed, either co-or posttranslationally, by the enzyme methionine aminopeptidase (MetAP) (1). In bacteria, this enzyme is the product of a single gene, and it is absolutely essential for bacterial survival, as demonstrated by gene deletion experiments in Escherichia coli (2) and Salmonella typhimurium (3). The single but critically essential MetAP enzyme of bacteria thus stands out as an attractive target for the design of antibacterial agents (4). Eukaryotes, on the other hand, have two distinct MetAPs, types I and II, arising from different genes (5). The human type II MetAP is a target of the antiangiogenic compounds fumagillin, ovalicin, and TNP-470 (6-8). Bengamides inhibit both types of MetAP (9) and cause inhibition of the growth of several human tumor cell lines in vitro at low-nanomolar concentrations. Therefore, human MetAPs may also serve as targets for the development of new anticancer agents. Some small molecules that inhibit MetAPs potently in vitro are known, but they lack potent antibacterial (10-12) or antiangiogenic (13) activities. One reason for this failure may be that they do not penetrate the bacterial or mammalian cells to...

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“…The down-regulation of MAP2 leads to the phosphorylation of eIF2a, resulting in the inhibition of protein synthesis, which ultimately triggers apoptosis (for review, see Datta, 2000). The C-terminal domain of animal MAP2s displays NME activity Yang et al, 2001). Natural compounds like fumagillin do not impair the interaction of MAP2 with eIF2a, but completely inhibit NME activity.…”

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confidence: 99%

Functional and Developmental Impact of Cytosolic Protein N-Terminal Methionine Excision in Arabidopsis

et al. 2005

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Protein N-terminal methionine (Met) excision (NME) is carried out by two types of Met aminopeptidases (MAPs), MAP1 and MAP2, in eukaryotes. Three enzymes, MAP1A, MAP2A, and MAP2B, have been identified in the cytoplasm of Arabidopsis (Arabidopsis thaliana). MAP transcript quantification revealed a predominance of MAP2B and developmental and organ-specific regulation of both MAP1A and MAP2s. By combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic NME in Arabidopsis and of the respective contributions of the two types of cytoplasmic MAPs throughout development. dsRNA interference and knockout (KO) plant lines targeting either MAP1A alone or both MAP2s simultaneously were constructed and shown to display wild-type phenotypes. In the MAP1A KO context, modulating MAP2 activity by treatment with various concentrations of the specific drug fumagillin impaired plant development, with particularly strong effects on the root system. Reciprocally, complete MAP2 inhibition in various MAP1A knocked-down genetic backgrounds also generated a gradient of developmentally abnormal plants, but the effects on the root system were milder than in the KO context. In the absence of MAP2 activity, the severity of the phenotype in the MAP1A knocked-down lines was correlated to the extent of MAP1A mRNA accumulation. Complete cytoplasmic NME inactivation blocked development after plant germination. Thus, in plants, (1) cytoplasmic NME is essential; (2) MAP1A and MAP2s are functionally interchangeable, which is not the case in fungi and animals, as a complete block of either MAP-type activity does not cause any visible molecular or phenotypic effect; and (3) a minimal level of cytoplasmic MAP is required for normal development.

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Steady-State Kinetic Characterization of Substrates and Metal-Ion Specificities of the Full-Length and N-Terminally Truncated Recombinant Human Methionine Aminopeptidases (Type 2)

Cited by 72 publications

References 41 publications

Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation

Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation

Structural basis of catalysis by monometalated methionine aminopeptidase

Functional and Developmental Impact of Cytosolic Protein N-Terminal Methionine Excision in Arabidopsis

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