Discovery, Identification, and Characterization of Candidate Pharmacodynamic Markers of Methionine Aminopeptidase-2 Inhibition

Abstract

The catalytic activity of methionine aminopeptidase-2 (MetAP2) has been pharmacologically linked to cell growth, angiogenesis, and tumor progression, making this an attractive target for cancer therapy. An assay for monitoring specific protein changes in response to MetAP2 inhibition, allowing pharmacokinetic (PK)/pharmacodynamic (PD) models to be established, could dramatically improve clinical decision-making. Candidate MetAP2-specific protein substrates were discovered from undigested cell culture-derived proteomes by MALDI-/SELDI-MS profiling and a biochemical method using (35)S-Met labeled protein lysates. Substrates were identified either as intact proteins by FT-ICR-MS or applying in-gel protease digestions followed by LC-MS/MS. The combination of these approaches led to the discovery of novel MetAP2-specific substrates including thioredoxin-1 (Trx-1), SH3 binding glutamic acid rich-like protein (SH3BGRL), and eukaryotic elongation factor-2 (eEF2). These studies also confirmed glyceraldehye 3-phosphate dehydrogenase (GAPDH) and cyclophillin A (CypA) as MetAP2 substrates. Additional data in support of these proteins as MetAP2-specific substrates were provided by in vitro MetAP1/MetAP2 enzyme assays with the corresponding N-terminal derived peptides and 1D/2D Western analyses of cellular and tissue lysates. FT-ICR-MS characterization of all intact species of the 18 kDa substrate, CypA, enabled a SELDI-MS cell-based assay to be developed for correlating N-terminal processing and inhibition of proliferation. The MetAP2-specific protein substrates discovered in this study have diverse properties that should facilitate the development of reagents for testing in preclinical and clinical environments.

Redox Regulation of Methionine Aminopeptidase 2 Activity

“…Cleavage of the initiating methionine in thioredoxin-1, eNOS, GAPDH, and cyclophilin A leads to N-terminal modification of the proteins at the second residue. Thioredoxin-1 is N-acetylated at Val 2 , eNOS is N-myristoylated at Gly 2 , GAPDH is N-acetylated at Gly 2 , and cyclophilin A is N-acetylated at Val 2 (11,32,33). These modifications can affect localization, activity, and turnover of these proteins and will be regulated by MetAP2 activity and, we suggest, the redox state of the allosteric disulfide.…”

“…The metalloproteases generally prefer amino acids with small and uncharged side chains at the second residue (P1Ј) of the sub-strate polypeptide chain. Alanine is preferred by MetAP1, whereas MetAP2 has a preference for valine, glycine, and cysteine (11). Six proteins have been identified as effectively specific MetAP2 substrates: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), thioredoxin, thioredoxin-like protein 1, Src homology 3 domain-binding glutamic acid-rich-like protein, elongation factor 2, and cyclophilin A (11,12).…”

“…Alanine is preferred by MetAP1, whereas MetAP2 has a preference for valine, glycine, and cysteine (11). Six proteins have been identified as effectively specific MetAP2 substrates: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), thioredoxin, thioredoxin-like protein 1, Src homology 3 domain-binding glutamic acid-rich-like protein, elongation factor 2, and cyclophilin A (11,12). The efficiency of cleavage of these substrates by MetAP2 is Ն200 times the cleavage by MetAP1.…”

Early Treatment with Fumagillin, an Inhibitor of Methionine Aminopeptidase-2, Prevents Pulmonary Hypertension in Monocrotaline-Injured Rats

“…How the inhibition of the enzymatic function of MetAP2 leads to p21 induction is not clear [33]. Incorrect processing of a protein's N-terminus can theoretically result in aberrant biological functions and lead to a cell stress response [45]. Although the current study has implicated the enzymatic activity of MetAP2 in promoting proliferation, MetAP2 has other recognized non-enzymatic functions.…”

Disruption of Wnt Planar Cell Polarity Signaling by Aberrant Accumulation of the MetAP-2 Substrate Rab37

“…Although several preferential MetAP-2 substrates (e.g., GAPDH, cyclophillin A and thioredoxin-1) have been identified, N-terminal Met retention on these proteins does not contribute to the effects of MetAP-2 inhibition on Wnt PCP signaling and angiogenesis (Wang, et al, 2008; Warder, et al, 2008). Here, using an unbiased N-terminal positional proteomics screen, we identify Rab37 as a preferential MetAP-2 substrate that aberrantly accumulates in the presence of TNP-470 and, when overexpressed, phenocopies the effects of MetAP-2 inhibition on Wnt PCP signaling-dependent processes such as endothelial cell proliferation/network formation and zebrafish embryogenesis.…”