Chapter 2

Methionine Aminopeptidase Emerging role in angiogenesis

Joseph A. Vetro1, Benjamin Dummitt2, and Yie-Hwa Chang2

1Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Ave., Lawrence, KS 66047, USA. 2Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.

Abstract: Angiogenesis, the formation of new blood vessels from existing vasculature, is a key factor in a number of vascular-related pathologies such as the metastasis and growth of solid tumors. Thus, the inhibition of angiogenesis has great potential as a therapeutic modality in the treatment of cancer and other vascular-related diseases. Recent evidence suggests that the inhibition of mammalian methionine aminopeptidase type 2 (MetAP2) catalytic activity in vascular endothelial cells plays an essential role in the pharmacological activity of the most potent small molecule angiogenesis inhibitors discovered to date, the fumagillin class. Methionine aminopeptidase (MetAP, EC 3.4.11.18) catalyzes the non-processive, co-translational hydrolysis of initiator N-terminal methionine when the second residue of the nascent polypeptide is small and uncharged. Initiator Met removal is a ubiquitous and essential modification. Indirect evidence suggests that removal of initiator Met by MetAP is important for the normal function of many proteins involved in DNA repair, signal transduction, cell transformation, secretory vesicle trafficking, and viral capsid assembly and infection. Currently, much effort is focused on understanding the essential nature of methionine aminopeptidase activity and elucidating the role of methionine aminopeptidase type 2 catalytic activity in angiogenesis. In this chapter, we give an overview of the MetAP proteins, outline the importance of initiator Met hydrolysis, and discuss the possible mechanism(s) through which MetAP2 inhibition by the fumagillin class of angiogenesis inhibitors leads to cytostatic growth arrest in vascular endothelial cells.

Key words: methionine aminopeptidase, angiogenesis, TNP-470, AGM-1470, ovalicin, vascular endothelial cells, protein turnover.

Aminopeptidases in Biology and Disease, Edited by Hooper and Lendeckel, Kluwer Academic/Plenum Publishers, New York, 2004 17

18 VETRO, DUMMITT, AND CHANG Chapter 2 1. CO-TRANSLATIONAL HYDROLYSIS OF INITIATOR METHIONINE

1.1 Overview

Eubacteria (Adams and Capecchi 1966; Clark and Marcker 1966), as well as mitochondria (Bianchetti et al. 1977) and chloroplasts (Lucchini and Bianchetti 1980) which are probably the descendants of endosymbiotic eubacteria (Gray 1992), initiate mRNA translation with an Nα-formylated methionine bound to an initiator tRNA (f-Met-tRNAf

Met) (Leder and Bursztyn 1966; Noll 1966). Eukaryotes (Housman et al. 1970) and archaebacteria (Ramesh and RajBhandary 2001) initiate the translation of cytosolic mRNA with a methionine-bound initiator tRNA (Met-tRNAi

Met). As a result, the primary structure of the majority of all nascent polypeptides, with the exception of a small number of proteins that initiate translation from rare non-AUG codons, begins with an initiating Nα-formylated N-terminal methionine (f-Metinit) or N-terminal methionine (Metinit).

Metinit is co-translationally hydrolyzed in a non-processive manner by methionine aminopeptidase (MetAP, E.C. 3.4.11.18, product of the MAP gene) when the second residue in the primary structure of the nascent polypeptide is small and uncharged (Ala, Cys, Gly, Pro, Ser, Thr, Val) (Adams 1968; Ben-Bassat et al. 1987; Boissel et al. 1985; Flinta et al. 1986; Huang et al. 1987; Tsunasawa et al. 1985). Metinit hydrolysis in eubacteria, however, only occurs after deformylation of f-Metinit (Adams 1968) as MetAP activity requires a free α-amino group (Solbiati et al. 1999). It is estimated that approximately 60 to 64% of mature proteins in Escherichia coli have Metinit removed (Hirel et al. 1989; Waller 1963). A similar survey in animal cells suggests that approximately 70% of mature proteins have Metinit removed (Boissel et al. 1985).

The hydrolysis of Metinit by MetAP is part of a series of possible co-translational N-terminal modifications (Figure 1). In eubacteria, the formyl group of f-Metinit is first removed from all nascent polypeptides by a peptide deformylase (PDF). Metinit is then hydrolyzed by MetAP when the nascent polypeptide is between 40 and 50 amino acids in length (Housman et al. 1972). In eukaryotes, Metinit is hydrolyzed when the nascent polypeptide is approximately 15 to 20 amino acids in length (Jackson and Hunter 1970). Metinit hydrolysis may then be followed by two secondary co-translational N-terminal modifications, Nα-acetylation (Ac) and Nα-myristoylation (Myr). These modifications occur when the nascent polypeptide is between 40 and 70 amino acids in the case of Nα-acetylation (Pestana and Pitot 1975a) or less than 100 amino acids in length in the case of Nα-myristoylation (Deichaite et

Methionine Aminopeptidase 19 al. 1988). Like the removal of Metinit by MetAP, the activities of the enzymes involved in these secondary modifications (Nα-myristoyltransferase [NMT] and Nα-acetyltransferase [NAT]) are predominantly governed by the primary structure of the nascent polypeptide, but additionally rely on the timely co-translational removal of Metinit to reveal the N-terminal substrate (Boutin 1997; Polevoda and Sherman 2000).

Figure 1. Summary of possible co-translational N-terminal modifications. Co-translational N-terminal modifications are defined as those that occur during polypeptide synthesis on the ribosomes and are largely governed by the primary sequence of the nascent polypeptide’s N-terminus (i.e., Metinit-AA2-AA3- where AAn is amino acid in position n). The minimal substrate requirements for the activity of each N-terminal modifying enzyme are summarized below the resulting N-terminal modification. Most mature proteins have some N-terminal modification.

1.2 Compartmentalization of Metinit and f-Metinit Hydrolysis in Higher Eukaryotes

It has been proposed that formylated Metinit is only hydrolyzed from Nα-formylated proteins synthesized in eubacteria and not from Nα-formylated proteins synthesized within mitochondria and chloroplasts (Mazel et al. 1994). This idea is based on the absence of a PDF gene in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans (Giglione et al. 2000b) as well as the genomes of mitochondria and chloroplasts. It was once

20 VETRO, DUMMITT, AND CHANG Chapter 2 assumed that, since eukaryotes do not possess a PDF, there would be no deformylation of f-Metinit and consequently no subsequent hydrolysis of Metinit by MetAP from proteins synthesized within mitochondria or chloroplasts (Giglione et al. 2000a). There is recent evidence, however, that both forms of Metinit hydrolysis are common in higher eukaryotes; Metinit hydrolysis takes place in the cytosol, and f-Metinit hydrolysis takes place in chloroplasts and mitochondria (Giglione et al. 2000b).

2. METHIONINE AMINOPEPTIDASES

2.1 MetAP Isoforms

Two major classes of MetAPs, designated type 1 and type 2 (MetAP1 and MetAP2), were originally identified as cytosolic proteins (Arfin et al. 1995; Li and Chang 1995). The two classes are structurally similar but share little sequence homology (Lowther and Matthews 2000). Eubacteria express only a type 1 MetAP (Chang et al. 1989), whereas archaebacteria express only a type 2 MetAP (Arfin et al. 1995; Li and Chang 1995). It has recently been shown, however, that the genome of the cyanobacterium, Synechocystis sp., has both MAP1 and MAP2 genes, as well as a novel MAP3 gene (Atanassova et al. 2003). Interestingly, all eukaryotes examined to date possess cytosolic forms of both MetAP1 and MetAP2 (Arfin et al. 1995; Li and Chang 1995). Multiple isoforms of MetAP1 were also recently found to localize to the chloroplasts (MetAP1B, MetAP1C, MetAP1D) and mitochondria (MetAP1C and MetAP1D) in the plant Arabidopsis thaliana (Giglione et al. 2000b). Although likely, it has not been demonstrated whether there are MetAP isoforms that localize to mitochondria of all eukaryotes. The distribution and variety of MetAPs are more complex than originally thought.

2.2 General Structural Features of MetAP

All MetAPs share a conserved C-terminal catalytic domain (Figure 2). Within the catalytic domain are five conserved amino acids that bind up to two metal ion cofactors. In Escherichia coli these five residues are Asp97, Asp108, His171, Glu204, and Glu235 (Roderick and Matthews 1993). Dialysis / flame emission analysis of MetAP1 from yeast (Saccharomyces cerevisiae) (Klinkenberg et al. 1997) and electron spin resonance analysis (Copik et al. 2003; D'souza et al. 2000) of MetAP from bacteria (Escherichia coli)

Methionine Aminopeptidase 21 suggest that the second metal ion binding site is only partially occupied in MetAP1.

X-ray crystal structure studies of MetAP from Escherichia coli (a type 1 MetAP) (Roderick and Matthews 1993), Pyrococcus furiosus (a type 2 MetAP) (Tahirov et al. 1998), and human MetAP2 (Liu et al. 1998) show that both type 1 and type 2 MetAPs exhibit a somewhat comparable “pita bread” symmetry (Roderick and Matthews 1993) in which both halves of the enzyme are structurally similar, although they share little sequence homology (for review see Lowther and Matthews 2000). A substrate binding pocket adjacent to the metal binding center capable of accommodating up to two amino acids has also been identified in both MetAP1 and MetAP2 (Liu et al. 1998; Roderick and Matthews 1993).

The major structural difference between the type 1 and type 2 MetAPs is an approximately 60 amino acid insert in the C-terminal catalytic domain of MetAP2 (Figure 2) (Arfin et al. 1995; Bazan et al. 1994; Li and Chang 1995). MetAP1 also has a narrower substrate binding pocket than MetAP2 (Liu et al. 1998; Tahirov et al. 1998).

Figure 2. Comparison of MetAP primary structures. Conserved metal binding residues within the C-terminal catalytic domain are shown (DDHEE).

Eukaryotic MetAPs, unlike bacterial MetAPs, additionally possess an extended N-terminal region that contains either a zinc finger domain (MetAP1) (Chang et al. 1992) or regions of charged residues (MetAP2) (Figure 2) (Arfin et al. 1995; Li and Chang 1995). Organellar MetAPs are

22 VETRO, DUMMITT, AND CHANG Chapter 2 more similar to bacterial MetAPs, but additionally possess mitochondrial or chloroplast targeting presequences in their N-terminal regions (Giglione et al. 2000b)

2.3 MetAP Cofactor in vivo

It was once assumed that cobalt is the metal ion cofactor of MetAP in vivo because the catalytic activity of purified MetAP is most effectively activated by cobalt in vitro. Recent studies of yeast MetAP1 activity in vitro, however, suggest that MetAP1 cannot bind cobalt in the presence of cellular concentrations of reduced glutathione (Walker and Bradshaw 1998). Among the metal cofactors tested under these conditions, zinc was found to be the least affected by glutathione. Based on these findings, it was proposed that MetAPs most likely utilize zinc as a cofactor in vivo (Walker and Bradshaw 1998). This hypothesis is consistent with the observation that cobalt-stimulated MetAP cannot hydrolyze Met-Cys- polypeptides (sulfhydryl-containing) in vitro, although Metinit is hydrolyzed from Met-Cys- proteins in vivo (Chen et al. 2002; Huang et al. 1987; Moerschell et al. 1990).

Alternatively, the total metal ion content of wild-type Escherichia coli compared to that of Escherichia coli overexpressing MetAP suggests that Fe (II) is the metal ion cofactor utilized by MetAP in vivo (D'souza and Holz 1999). It has been suggested that these studies may only reflect the uptake of the most abundant metal cation in the host cells and not the physiologically relevant cofactor at the normal steady state levels of MetAP protein (Yang et al. 2001). Whether proteins use the most available metal ion cofactors or only specific metal ions in vivo, however, remains unclear.

Recent studies using selective inhibitors of MetAP2 bound with various cofactors have also demonstrated that manganese is the cofactor for mammalian MetAP2 in vivo (Wang et al. 2003). Thus, the identity or identities of the actual cofactor utilized by MetAPs in vivo remains unresolved.

2.4 Eukaryotic MetAPs

2.4.1 Eukaryotic MetAP1

In addition to the conserved C-terminal catalytic domain, eukaryotic MetAP1 possesses an extended N-terminal region (Figure 2). Within this N-terminal region, eukaryotic MetAP1 has a zinc finger domain (Chang et al. 1992) consisting of a Cys2-Cys2 zinc finger motif similar to the RING finger family and a unique Cys2-His2 zinc finger motif similar to zinc fingers

Methionine Aminopeptidase 23 involved in RNA binding (Zuo et al. 1995). Furthermore, yeast MetAP1 chelates zinc in a 2:1 molar ratio of zinc:MetAP1, consistent with the presence of two zinc finger motifs, and deletion of the zinc finger domain abrogates zinc binding (Zuo et al. 1995).

MetAP1 is primarily responsible for cellular Metinit hydrolysis activity in yeast (Vetro and Chang 2002). Although not sufficiently investigated, preliminary evidence suggests that mammalian MetAP1 is also predominantly responsible for cellular Metinit hydrolysis activity in animal cells (Turk et al. 1999).

2.4.2 Subcellular distribution of yeast MetAP1

We recently reported direct biochemical evidence that yeast MetAP1 is ribosome associated, consistent with earlier evidence suggesting that eukaryotic MetAPs are localized to the ribosomes (Jackson and Hunter 1970). Ribosome profile experiments in yeast revealed that the majority of MetAP1 is associated with the 60S ribosome subunit and suggest that MetAP1 is recruited from the cytosol to the 80S translational complex through this association (Vetro and Chang 2002). The MetAP1 ribosome association was also found to be sensitive to concentrations of Mg2+ greater than 2 mM (Vetro and Chang 2002) and can occur in the absence of MetAP2 (Vetro, unpublished data). The presence of a polypeptide channel in the 60S ribosome subunit (Morgan et al. 2000) further suggests that MetAP1 may be localized to the 60S subunit near the exit of the polypeptide channel where it can co-translationally hydrolyze Metinit from nascent polypeptides in a timely manner (Vetro and Chang 2002; Zuo et al. 1995). The subcellular distribution of mammalian MetAP1 remains to be determined.

2.4.3 Possible role of the zinc finger domain in yeast MetAP1 ribosome association

Although not required for catalytic activity in vitro, we have reported genetic evidence indicating that the zinc finger domain is required for the normal function of yeast MetAP1 in vivo (Zuo et al. 1995). A comparison of the ribosome profile distributions of wild-type yeast MetAP1 and truncated MetAP1 lacking the entire zinc finger domain with their individual Metinit hydrolysis efficiencies in vivo suggests that the zinc finger domain maintains the correct functional alignment of MetAP1 on the ribosomes. A similar analysis of yeast MetAP1 zinc finger point mutants further suggests that ribosome association is important for normal MetAP1 function in vivo (Vetro and Chang 2002).

24 VETRO, DUMMITT, AND CHANG Chapter 2 2.5 Eukaryotic MetAP2

In addition to the conserved C-terminal catalytic domain, eukaryotic MetAP2, like eukaryotic MetAP1, possesses an extended N-terminal region (Figure 2). Within this N-terminal region, eukaryotic MetAP2 has a single polylysine block (yeast MetAP2) (Arfin et al. 1995; Li and Chang 1995) or a polyaspartate block flanked by two polylysine blocks (mammalian MetAP2 / rat p67) (Wu et al. 1993). Mammalian MetAP2 is additionally post-translationally modified with O-linked N-acetylglucosamine moieties that appear to play a role in the subcellular distribution and regulation of MetAP2 in animal cells (Datta 2000).

2.5.1 Subcellular distribution of MetAP2

Ribosome profile experiments revealed that, in addition to being a cytosolic protein, yeast MetAP2 associates with the 40S and 60S ribosome subunits and 80S translational complex (Vetro, unpublished data). In the profile, distribution of yeast MetAP2 was found to parallel the distribution of yeast MetAP1 (Vetro, unpublished data). Furthermore, the association of yeast MetAP2 with the ribosomes was also found to be sensitive to concentrations of Mg2+ greater than 2 mM and does not require the presence of yeast MetAP1 (Vetro, unpublished data). It remains unclear, however, whether ribosome association is required for the normal function of yeast MetAP2 in vivo.

The subcellular distribution of MetAP2 in animal cells appears to be more complicated than in yeast. Mammalian MetAP2 (rat p67) was first characterized as a 67-kDa protein involved in positively regulating protein synthesis by associating with eukaryotic initiation factor 2 (eIF2) (Datta et al. 1988) and consequently blocking eIF2 phosphorylation (for review see Wek 1994). It was recently shown that MetAP2 associates with the metastasis association protein S100A4 in a Ca++-dependent manner (Endo et al. 2002). A yeast two-hybrid screen also identified the caveolae-associated protein flotillin as a MetAP2 interacting protein (Liu and Liu 2001). Thus, mammalian MetAP2 may be additionally regulated by shuttling among various associations within the cell. It has not been determined if mammalian MetAP2 is associated with ribosomes.

2.5.2 Role of the N-terminal region in yeast MetAP2 function

Unlike wild-type yeast MetAP2 (Li and Chang, 1995), overexpression of truncated MetAP2 (∆2-57), which lacks the entire polylysine block, cannot rescue the slow-growth phenotype of a yeast map1∆ strain (Vetro,

Methionine Aminopeptidase 25 unpublished data). Although the catalytic activity of yeast MetAP2 (∆2-57) was not determined, N-terminal truncation does not affect the catalytic activity of yeast MetAP1 (68 residues) (Zuo et al. 1995) or human MetAP2 (107 residues; hMetAP2) (Yang et al. 2001) in vitro. Furthermore, a comparison between the CD spectra of truncated hMetAP2 (∆2-108) and full-length hMetAP2 indicates that N-terminal truncation does not significantly alter the secondary structure of hMetAP2 (Yang et al. 2001). Together, these results suggest that residues 2-57 are not required for yeast MetAP2 catalytic activity, but, like yeast MetAP1, are essential for normal function in vivo.

Interestingly, N-terminal residues 2-57 are also required for the dominant negative activity of a catalytically inactive mutant of yeast MetAP2 (Vetro et al., submitted). Thus, residues 2-57 may be important for the association of MetAP2 with an unidentified cytosolic factor that is required for normal function in vivo. Polylysine blocks have been found to mediate protein-protein and protein-nucleic acid interactions in other proteins involved in mRNA translation such as human eIF2-β (Asano et al. 1999), its yeast homologue, Sui3 (Pathak et al. 1988), elongation factor-3 (EF-3) (Chakraburtty 1999), and human Nα-myristoyltransferase (hNMT) (Glover et al. 1997).

2.5.3 Regulation of mammalian MetAP2

Mammalian MetAP2 expression is regulated both transcriptionally and post-translationally. Transcription of rat MetAP2 (p67) is induced by phorbol 13-myristate 12-acetate (PMA) (Chatterjee et al. 1997). Additionally, p67 is absent from serum-starved rat KRC-7 cells, and treatment with p67 antisense DNA prevents PMA induction of p67 and subsequent protein synthesis (Gupta et al. 1997), which demonstrates the importance of p67 in growth and protein synthesis. Deglycosylation followed by degradation of p67 also occurs in heme-deficient reticulocyte lysates and in serum-starved KRC-7 cells in culture (Ray et al. 1992). Whether mammalian MetAP2 is universally regulated in this manner remains to be determined.

2.6 MetAP Substrate Specificity

Studies of proteins with mutated N-termini and extensive database searches of mature proteins initially suggested that the second residue of a protein predominantly governs Metinit hydrolysis activity, as the second residue is often found to be Ala, Cys, Gly, Pro, Ser, Thr, or Val (Boissel et al. 1985; Flinta et al. 1986; Tsunasawa et al. 1985). The activities of purified MetAP1 from Escherichia coli (Ben-Bassat et al. 1987), Salmonella

26 VETRO, DUMMITT, AND CHANG Chapter 2 typhimurium (Miller et al. 1987), and Saccharomyces cerevisiae (Chang et al. 1990) and purified MetAP2 from pig (Kendall and Bradshaw 1992), Saccharomyces cerevisiae (Li and Chang 1995), and human (Li and Chang 1996) against short peptide substrates in vitro confirmed this specificity.

Although the second residue primarily determines if Metinit hydrolysis occurs, differences between the activities of MetAP1 and MetAP2 against the same protein substrate have been observed in vivo (Chen et al. 2002; Towbin et al. 2003; Turk et al. 1999). There are currently at least two possible reasons for differences in MetAP1 and MetAP2 activities in vivo. It has been shown that an increase in peptide length increases MetAP catalytic activity in vitro (Chang et al. 1990) and results in a decrease in the Km of the polypeptide substrate (Walker and Bradshaw 1999). The increased affinity for longer polypeptides was suggested to occur primarily through peptide backbone interactions with MetAP that are largely sequence-independent (Walker and Bradshaw 1999). Recent studies, however, show that human MetAP1 and MetAP2 have different activities in vitro against octapeptide substrates with the same second residue but different downstream residues (Turk et al. 1999). These results indicate that peptide interactions with MetAP do depend on residues downstream of the second residue and may account for differences between the specificities of MetAP1 and MetAP2 in vivo (Turk et al. 1999).

A second possibility for differences in the activities of MetAP1 and MetAP2 against specific substrates is that MetAP2 catalytic activity is sensitive to product inhibition by cytosolic concentrations of methionine. The IC50 for methionine inhibition of human MetAP2 (150 µM) was recently found to be at least 30 times less than that for human MetAP1 (>5 mM) (Dummitt et al. 2003). Furthermore, normal expression levels of MetAP2 are insufficient for the cellular MetAP activity requirements of yeast (Chen et al. 2002). Thus, it is likely that methionine sensitivity and relative expression levels additionally affect MetAP2 activity in vivo.

3. IMPORTANCE OF METinit HYDROLYSIS

Metinit hydrolysis is an essential cellular function. Deletion of the single MAP gene in Escherichia coli (Chang et al. 1989) and Salmonella typhimurium (described as pepM [peptidase M]) (Miller et al. 1989) or of both MAP genes (MAP1 and MAP2) in the yeast Saccharomyces cerevisiae is lethal (Li and Chang 1995). Furthermore, treatment of both a primary and immortalized human cell line with LAF389, an analog of bengamide B found to reversibly inhibit MetAP1 and MetAP2 catalytic activities with similar potency, causes extensive cell death at concentrations that

Methionine Aminopeptidase 27 significantly inhibit the activities of both MetAPs in vitro (Towbin et al. 2003).

In contrast, deletion of MAP1 or MAP2 alone in yeast results in a slow-growth phenotype (Chang et al. 1992; Li and Chang 1995). The slow-growth phenotype of the yeast map1 knockout strain (map1∆), however, is more severe than the yeast map2 knockout strain (map2∆) (Chang et al. 1992; Li and Chang 1995). This result is consistent with MetAP1 being primarily responsible for cellular Metinit hydrolysis activity in yeast (Chen et al. 2002). MetAP2, on the other hand, is likely to be important for the proliferation of endothelial cells, P. falciparum and Leishmania donavani (Sin et al. 1997; Griffith et al. 1997; Zhang et al. 2002).

3.1 Possible Ways that Metinit Retention Affects Cell Viability

Although the absence of cellular MetAP activity is lethal, the mechanism(s) responsible for lethality remain unclear. Considering that Met is the most energetically expensive amino acid to synthesize in lower organisms, Metinit hydrolysis probably evolved as part of a salvage pathway to partially recover Met that would normally be sequestered on the N-terminus of every protein (Meinnel et al. 1993). This would ensure that translation initiation is not abnormally interrupted and help to maintain cytosolic levels of Met for other Met-dependent metabolic pathways. Thus, one possibility is that extensive Metinit retention decreases cytosolic levels of Met and consequently interferes with protein synthesis and/or Met-dependent metabolism. We recently demonstrated that deletion of MAP1 in yeast results in the upregulation of methionine biosynthetic genes, thus confirming the importance of methionine removal in maintaining cellular Met pools (Dummitt et al. 2003). However, the fact that lower organisms like yeast can synthesize Met but cannot survive in the absence of MetAP activity suggests that separate or additional mechanisms likely contribute to lethality.

There is much indirect evidence suggesting that many proteins rely on Metinit hydrolysis for normal function in vivo. Thus, another possibility is that the absence of MetAP activity results in the dysfunction of one or more MetAP substrate proteins that are required for cell viability and/or cell growth. Currently, proteins that probably require Metinit hydrolysis for normal function in vivo can be categorized into two groups (Zuo et al. 1995). The first group of proteins requires Metinit hydrolysis for the remaining N-terminal residues to function normally in catalysis or allosteric regulation. For example, T4 endonuclease V requires Metinit hydrolysis to expose the catalytic α-amino group of the second residue (threonine) (Schrock and

28 VETRO, DUMMITT, AND CHANG Chapter 2 Lloyd 1993). This is thought to give the α-amino group sufficient proximity to the substrate for normal catalytic function. Glutamine phosphoribosyl- pyrophosphate (PRPP) amidotransferase also requires Metinit hydrolysis to expose the α-amino group of the second residue Cys to participate in catalysis (Smith 1998), whereas the β subunits of hemoglobin require Metinit hydrolysis for normal allosteric properties (Barwick et al. 1985).

The second group of proteins relies indirectly on Metinit hydrolysis to allow subsequent co-translational N-terminal modifications to occur that are required for normal protein function in vivo (e.g., Nα-myristoylation, Nα-acetylation). The first N-terminal modification, Nα-myristoylation, is a ubiquitous and essential modification found only on eukaryotic and viral proteins (for review see Boutin 1997). Nα-myristoyltransferase (NMT) co-translationally transfers the fatty acid myristate from myristoyl coenzyme A to N-terminal glycine. This transfer is governed by consensus sequences in the primary structure of a protein’s N-terminus (Figure 1) (Boutin 1997; Utsumi et al. 2001; Wilcox et al. 1987). NMT, however, additionally requires the hydrolysis of Metinit in order to expose the α-amino group of N-terminal glycine for modification (Towler et al. 1987).

Several eukaryotic proteins require Nα-myristoylation for normal function in vivo. Examples include many proteins involved in cell signaling such as the catalytic subunit of the cAMP-dependent protein kinase (PKA) (Raju et al. 1997) and the α-subunits of several G proteins (Gordon et al. 1991). Proteins involved in the regulation of protein secretory vesicular trafficking through the Golgi stacks (e.g., Arf1p) also require Nα-myristoylation for proper function in vivo (Kahn et al. 1995).

The second co-translational N-terminal modification, Nα-acetylation, with very few exceptions, is found only in eukaryotes and relies almost entirely on Metinit hydrolysis before it can occur in vivo (for review see Polevoda and Sherman 2000). Nα-acetyltransferase (NAT) co-translationally transfers an acetate group from acetyl-CoA to the α-amino group of N-terminal glycine, alanine, serine, and threonine (GAST substrates), as well as methionine, on nascent polypeptides 40 to 70 amino acids in length (Figure 1) (Pestana and Pitot 1975b). Three different forms of NAT have been identified (NATA, NATB, NATC), and each is a complex made up of a combination of the catalytic subunits Ard1p, Nat3p, and Mak3p (Polevoda and Sherman 2000). Each NAT complex modifies a specific subset of proteins having different N-terminal regions (Polevoda and Sherman 2000).

The biological significance of Nα-acetylation is less understood than Nα-myristoylation. Deletion of nat1 and ard1 causes a slow-growth phenotype in yeast, presumably due to the absence of Nα-acetylation on a subset of proteins that require it for normal function in vivo and are modified only by NAT complexes formed from nat1 and ard1 (Polevoda and Sherman 2000).

Methionine Aminopeptidase 29 Two clear examples of the importance of Nα-acetylation are the increased melanotropic effects of α-melanocyte-stimulating hormone and the reduced analgesic action of β-endorphin after Nα-acetylation (Polevoda and Sherman 2000).

In contrast to abnormal Metinit retention, there is only one example to date where the abnormal hydrolysis of Metinit causes dysfunction in vivo. When Metinit is hydrolyzed from rat liver mitochondrial aldehyde dehydrogenase because of a Leu to Val mutation in the second residue, the protein is no longer translocated to the mitochondria (Hammen et al. 1999). It is proposed that the abnormal hydrolysis of methionine decreases the hydrophobic surface area of the N-terminal region, which is required for transport by the mitochondrial import machinery (Hammen et al. 1999).

3.2 The Role of MetAP in Protein Turnover

The N-end rule states that the intracellular half-life of a protein is governed by its N-terminal residue (for review see Varshavsky 1997). Proteins with destabilizing N-terminal residues (e.g., Arg, Phe) have short half-lives in vivo, whereas proteins with stabilizing N-terminal residues (e.g., Met, Ser) have considerably longer half-lives in vivo (Varshavsky 1997). Methionine is considered a stabilizing N-terminal residue and proteins that retain Metinit generally have long half-lives in vivo (Varshavsky 1997). Considering that Metinit hydrolysis by MetAP in vivo almost always reveals a stabilizing residue (the role of N-terminal Pro in protein turnover is unclear) (Bachmair et al. 1986), it seems unlikely that MetAP, in general, is directly involved in regulating protein turnover. However, one example that indirectly requires Metinit hydrolysis is the degradation of proteins mediated by the mammalian Arg-tRNA-transferase (R-transferase), ATE-1. ATE-1 targets specific proteins for degradation through the N-end rule pathway by transferring a destabilizing Arg to the N-terminus of the protein (Gonda et al. 1989). One of the potential substrates of ATE-1, N-terminal Cys, requires Metinit hydrolysis from Met-Cys- and subsequent oxidation of Cys before ATE-1 can transfer Arg to the N-terminus of the protein (Kwon et al. 2002). Retention of Metinit on Met-Cys- proteins consequently blocks R-transferase activity and likely stabilizes proteins targeted by ATE-1. This branch of the N-end rule pathway, however, relies directly on the activation and specificity of ATE-1 as Metinit hydrolysis from Met-Cys- substrates by MetAP alone will not target proteins for degradation.

MetAPs may also be involved in protein turnover in specific cases that are not readily predictable. Using a recombinant mutant glutathione-S-transferase protein, we recently found evidence that the abnormal retention of Metinit may increase a protein’s turnover rate in yeast (Chen et al. 2002).

30 VETRO, DUMMITT, AND CHANG Chapter 2 Additionally, it has been shown that PDF inhibitors can destabilize photosystem 2 components in Chlamydomonas reinhardtii cells and that this effect results from the subsequent retention of initiator methionine (Giglione et al. 2003). Although the physiological relevance of these findings is unclear, abnormal protein turnover resulting from Metinit retention may have important consequences under pathological or pharmacological conditions where cellular Metinit hydrolysis activity is inhibited.

4. ROLE OF MAMMALIAN METAP2 CATALYTIC ACTIVITY IN THE PHARMACOLOGICAL MECHANISM OF THE FUMAGILLIN CLASS OF ANGIOGENESIS INHIBITORS

4.1 Angiogenesis

Blood vessel growth and formation from existing vasculature (angiogenesis) is a normal, physiological process that occurs during embryonic development, the female reproductive cycle, and wound repair (Fan et al. 1995). Angiogenesis involves a complex mechanism of growth factor-mediated activation, migration, and proliferation of both vascular endothelial cells (VEC) and smooth muscle cells (SMC) as well as rearrangement of the local extracellular matrix (Amant et al. 1999; Carmeliet and Collen 1997). The entire process leads to the formation of a structurally competent vessel wall composed of an inner layer of VEC and an outer layer of SMC (Carmeliet and Collen 1997).

Under normal conditions, angiogenesis is a controlled and highly regulated process (Fan et al. 1995). Under pathological conditions, however, angiogenesis can be excessive (e.g., diabetic retinopathy, neoplasia) or inadequate (e.g., lower limb ischemia, myocardial ischemia, ischemic ulcerations). Angiogenesis is a key factor in a number of vascular and vascular-related diseases (for review see Folkman 1997). Thus, the modulation of angiogenesis could be potentially beneficial in the treatment of these diseases. This is an especially promising modality in the treatment of cancer where angiogenesis is an essential factor in tumor growth and metastasis (Folkman 1997).

The fumagillin class of anti-angiogenesis antibiotics (“angioinhibins” Ingber et al. 1990) which are either analogues (e.g., TNP-470) or structurally similar (e.g., ovalicin, isolated from Pseudorotium ovalis (Sigg and Weber 1968) to the naturally occurring antibiotic fumagillin (Figure 3), are the most potent natural angiogenesis inhibitors discovered to date (Figure 3) (for

Methionine Aminopeptidase 31 review see Kruger and Figg 2000). Fumagillin was originally characterized as an antibiotic secreted from the fungus Aspergillus fumigatus fresenius, and it inhibits angiogenesis, in part, by causing growth arrest in VECs (Ingber et al. 1990). Ovalicin is a naturally-occuring analogue isolated from Pseudorotium ovalis (Sigg and Weber 1968). A less toxic derivative of fumagillin, TNP-470 (previously AGM-1470), is 50 times more potent than fumagillin at inhibiting VEC growth in vitro (Ingber et al. 1990) and is the first anti-angiogenic compound to enter clinical trials (Kruger and Figg 2000). Consequently, much of the work to date has focused on TNP-470.

Figure 3. The Fumagillin class of angiogenesis inhibitors. Carbon numbering was adopted from Liu et al. 1998

4.2 Targeting of Mammalian MetAP2 by the Fumagillin Angioinhibins

Evidence that fumagillin and ovalicin target and irreversibly inhibit mammalian MetAP2 catalytic activity in VECs was recently reported. Near

32 VETRO, DUMMITT, AND CHANG Chapter 2 western analyses of crude protein extracts from primary VECs using biotin-labeled fumagillin (Sin et al. 1997) or photoaffinity-labeled ovalicin (Griffith et al. 1997) showed a preferentially labeled 67-kDa protein that was identified as MetAP2. Furthermore, concentrations of TNP-470 and ovalicin up to 10 µM have no effect on yeast MetAP1 catalytic activity in vitro, whereas yeast MetAP2 catalytic activity is completely inhibited at concentrations as low as 5 nM (Griffith et al. 1997). Thus, TNP-470 and ovalicin have a high specificity for MetAP2.

Crystal structure studies of human MetAP2 complexed with fumagillin revealed that the imidazole nitrogen (Nε2) of the conserved catalytic residue His231 covalently attaches to the C3 ring epoxide group of fumagillin (Liu et al. 1998). These studies are further supported by the findings that the substitution of His231 with Asn in hMetAP2 precludes the covalent attachment of fumagillin to hMetAP2 and that the C3 ring epoxide group is required for EC growth inhibition by the fumagillin class of angiogenesis inhibitors (Griffith et al. 1998). Differences between the geometries of the MetAP1 and MetAP2 substrate binding pockets, which do not favor fumagillin adduct formation with MetAP1, likely account for fumagillin’s specificity for MetAP2 (Liu et al. 1998).

4.3 The Inhibition of VEC Growth by TNP-470

TNP-470 exhibits a biphasic inhibition of VEC growth in vitro (Kusaka et al. 1994). At concentrations ranging from 740 pM to 7.4 µM, treatment of human umbilical vein endothelial cells (HUVECs) with TNP-470 results in a complete and reversible cytostatic growth arrest (IC50 37 pM) (Kusaka et al. 1994). Similar growth sensitivity to low concentrations of TNP-470 is observed in human embryonic lung fibroblasts and rat SMCs (Kusaka et al. 1994), whereas other cell types are relatively unaffected at the same concentrations (Kusaka et al. 1994; Wang et al. 2000). Thus, growth sensitivity to TNP-470 treatment is cell type-specific (Kusaka et al. 1994). The growth of endothelial cells, however, appears to be the most sensitive (Kusaka et al. 1994).

Based on genetic evidence suggesting that cellular levels of Metinit hydrolysis activity directly correlate with growth rate in yeast (Chang et al. 1992), it has been proposed that differences in the combined expression of MetAP1 and MetAP2 among cell types may account for differences in growth sensitivity to TNP-470 (Sin et al. 1997). The combined expression levels of MetAP1 and MetAP2 in sensitive versus non-sensitive cell types, however, do not correlate with sensitivity to TNP-470 (Turk et al. 1999; Wang et al. 2000; Yeh et al. 2000).

Methionine Aminopeptidase 33

At concentrations of TNP-470 greater than ~25 µM, HUVEC growth inhibition is irreversible, whereas concentrations equal to or exceeding 74 µM are cytotoxic (Kusaka et al. 1994). Unlike cytostatic growth arrest, which affects only certain cell types, high concentrations of TNP-470 are cytotoxic to all cell types examined to date (Kusaka et al. 1994). The cytotoxicity of TNP-470 may be a consequence of inhibiting the Metinit hydrolysis activities of both MetAP1 and MetAP2, analogous to the lethality observed when both MAP1 and MAP2 are deleted in yeast (Li and Chang 1995). This proposal is further supported by the finding that high concentrations of ovalicin (structurally similar to TNP-470) covalently modify Escherichia coli MetAP (a type 1 MetAP) and yeast MetAP1 in the same manner as hMetAP2 (Lowther et al. 1998).

4.3.1 TNP-470 inhibition of VEC growth through inhibition of MetAP2 catalytic activity

There is much evidence indicating that the selective, cytostatic inhibition of VEC growth is responsible for the anti-angiogenic activity of TNP-470 (Kusaka et al. 1994). It has been further proposed that the inhibition of MetAP2 catalytic activity by TNP-470 is responsible for cytostatic growth arrest in VEC (Griffith et al. 1997; Sin et al. 1997; Turk et al. 1999) and is supported by a comparison of fumagillin and ovalicin analogues which shows a correlation (P > 0.001) between the inhibition of MetAP2 activity and the inhibition of EC growth in vitro (Griffith et al. 1997). Furthermore, the concentration of TNP-470 sufficient to modify half of MetAP2 in intact EC (100 ± 13 pM) is comparable to the IC50 of TNP-470 that is cytostatic under the same conditions (161 ± 69 pM) (Turk et al. 1999). Rationally designed drugs based on the crystal structure of hMetAP2 also show much greater potency against EC growth (Han et al. 2000; Wang et al. 2003). The recent development of a more chemically stable, reversible inhibitor of MetAP2 further demonstrates that MetAP2 is a physiologically relevant target for VEC growth inhibition in vivo (Wang et al. 2003).

It is possible that TNP-470 additionally inhibits EC growth by disrupting the role of MetAP2 in protein synthesis regulation. This is unlikely, however, because total protein synthesis in HUVECs is unaffected at cytostatic concentrations of TNP-470 (Kusaka et al. 1994) and because steady state levels of MetAP2 actually increase in response to TNP-470 (Wang et al. 2000). Furthermore, TNP-470-bound MetAP2 is as effective as unbound MetAP2 in blocking eIF2α phosphorylation by heme-regulated kinase (Griffith et al. 1997). We also have preliminary evidence that a dominant negative mutant of hMetAP2 inhibits endogenous MetAP2 activity as well as HUVEC growth (Ying Fei, unpublished data). Together, these

34 VETRO, DUMMITT, AND CHANG Chapter 2 observations indicate that TNP-470 does not interfere with the role of MetAP2 (p67) in regulation of protein synthesis and strongly support the proposal that the inhibition of MetAP2 catalytic activity by TNP-470 is an essential step in the cytostatic inhibition of VEC growth (Griffith et al. 1997; Sin et al. 1997; Turk et al. 1999).

4.3.2 Mechanism of cytostatic growth arrest by TNP-470

Early studies suggested that TNP-470 blocks S-phase entry in ECs by interfering with mitogenic signaling in the G1-phase (Kusaka et al. 1994). An important downstream factor of G1-phase mitogenic signaling is the gene regulatory protein, E2F (for review see Chan et al. 2001). E2F promotes the transcription of genes required for S-phase entry and is regulated by an association with the hypophosphorylated form of retinoblastoma protein (Rb), which prevents S-phase entry by preventing the transactivation function of E2F. Initial hyperphosphorylation of Rb by G1-phase cyclin-dependent kinase complexes (cyclin D-cdk4 and cyclin D-cdk6) in response to mitogenic signaling releases E2F from Rb and leads to the downstream activation of the G1/S-phase Rb kinase cyclin E-cdk2 and the S-phase Rb kinase cyclin A-cdk2. Cyclin E-cdk2 then phosphorylates additional Rb as well as the cyclin A-cdk2 regulatory factors, Sic-1 and Hct-1, to help activate cyclin A-cdk2 and drive S-phase entry.

Recent studies show that TNP-470 blocks S-phase entry in ECs by inhibiting Rb hyperphosphorylation through the activation of the p53 pathway (Figure 4). Treatment of primary ECs with cytostatic concentrations of TNP-470 inhibits cyclin E-cdk2 activity and subsequent Rb hyperphosphorylation through the p53-dependent induction of p21WAF1/CIP1 (Yeh et al. 2000; Zhang et al. 2000).

4.3.3 Evidence that TNP-470 activates the p53 pathway by a unique mechanism in endothelial cells

Although p53 and p21WAF1/CIP1 are required for growth arrest of ECs by TNP-470, the cellular events that lead to p53 activation remain unclear. TNP-470 treatment increases the steady state level of p53 protein in HUVECs, but has no effect on the steady state level of p53 mRNA (Zhang et al. 2000). This indicates that TNP-470 increases p53 steady state levels through a post-transcriptional mechanism (Zhang et al. 2000). Furthermore, it is unlikely that p53 is activated through a general genotoxic mechanism such as DNA damage because the growth of primary murine adult lung fibroblasts is only 20% inhibited by concentrations of TNP-470 that inhibit 90% of VEC growth (Yeh et al. 2000).

Methionine Aminopeptidase 35

Both the activity and protein levels of p53 are downregulated by the p53-dependent expression of Mdm2 (Agarwal et al. 1998). Mdm2 is an ubiquitin ligase that associates with p53 to block p53-dependent transcription and targets p53 for degradation by the proteasome (Agarwal et al. 1998). Thus, an increase in p53 steady state levels could occur by blocking the association of Mdm2 with p53.

Figure 4. Potential mechanism(s) of VEC cytostatic growth arrest after TNP-470 treatment.

Phosphorylation of p53 has been shown to decrease the association of p53 and Mdm2 (Agarwal et al. 1998). However, Mdm2 expression increases in primary murine pulmonary endothelial cells (MPEs) after TNP-470

36 VETRO, DUMMITT, AND CHANG Chapter 2 treatment (Yeh et al. 2000), and the phosphorylation of common sites on p53 (Ser-6, -9, -15, -20, -37, and -392) does not occur (Yeh et al. 2000). These results suggest that phosphorylation of p53 is not involved in interfering with the regulation of p53 by Mdm2 after TNP-470 treatment (Yeh et al. 2000). It remains possible, however, that other sites are phosphorylated on p53 that interfere with Mdm2 association (Yeh et al. 2000).

A second protein, p19ARF, increases p53 steady state levels by binding Mdm2 and sequestering it in the nucleolus as well as targeting Mdm2 for proteasome degradation (Agarwal et al. 1998). Nucleolar localization of epitope-tagged Mdm2 or GFP-p19ARF, however, is not observed in wild-type MPEs after TNP-470 treatment (Yeh et al. 2000). Together, these preliminary results suggest that TNP-470 treatment activates the p53 pathway through a unique mechanism in ECs (Yeh et al. 2000; Zhang et al. 2000).

4.3.4 Possible downstream effects of MetAP2 inhibition that lead to p53 activation

Given the specificity of TNP-470 for MetAP2, it has been proposed that differences in substrate processing efficiencies between MetAP1 and MetAP2 in vivo might lead to the retention of Metinit on proteins that rely primarily on MetAP2 for Metinit hydrolysis (Griffith et al. 1997; Sin et al. 1997; Turk et al. 1999). The retention of Metinit may then interfere with the function of one or more of these MetAP2-reliant proteins, thus resulting in the activation of the p53 pathway and subsequent cell cycle arrest (Figure 4).

Evidence of selective Metinit retention on VEC proteins after treatment with cytostatic concentrations of TNP-470 has been reported. A 2-D gel comparison of pulse-labeled proteins from treated and untreated bovine aortic endothelial cells (BAEC) revealed several proteins with abnormal migrations (Turk et al. 1999). One of the abnormally migrating proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which begins with Met-Val), was found to retain ~70% of Metinit, whereas the extent of Metinit removal of another protein, glutathione S-transferase-π (GST-π, which begins with Met-Pro-), was not affected (Turk et al. 1999). Thus, although both GAPDH and GST-π are MetAP substrates, only GAPDH is affected by MetAP2 inhibition. GAPDH, however, is unlikely to be involved in the activation of the p53 pathway (Turk et al. 1999). Additional abnormally migrating proteins were recently identified in HUVECs after treatment with a reversible inhibitor of both MetAP1 and MetAP2, but it remains undetermined whether they are involved in p53 activation (Towbin et al 2003).

Methionine Aminopeptidase 37

Considering that Nα-myristoylation is required for the function of many signalling proteins and that NMT requires Metinit hydrolysis to reveal N-terminal glycine substrates (Towler et al. 1987), it is possible that TNP-470 treatment interferes with Nα-myristoylation of MetAP2-reliant signalling proteins in VECs (Griffith et al. 1997; Sin et al. 1997). Studies in BAECs showed that TNP-470 treatment decreases eNOS Nα-myristoylation and concomitantly increases the cytosolic concentrations of eNOS (Yoshida et al. 1998), presumably due to decreased eNOS Nα-myristoylation, which is required for translocation to the caveolae of the plasma membrane (Busconi and Michel 1993). An increase in intracellular concentrations of NO was also observed after TNP-470 treatment and likely results from the abnormal localization of eNOS in the cytosol (Busconi and Michel 1993). Given that reactive oxygen species (ROS) such as NO can activate the p53 pathway through DNA damage and/or mitochondrial dysfunction and cause growth arrest or apoptosis (Agarwal et al. 1998), it was proposed that TNP-470 blocks angiogenesis, in part, by inducing apoptosis in VEC (Yoshida et al. 1998). The concentrations of TNP-470 used in these studies (1.2 µM to 5 µM), however, are at least 1000-fold greater than the concentration of TNP-470 that is completely cytostatic to VEC in vitro (~1 nM) (Kusaka et al. 1994). Furthermore, treatment of VEC with cytostatic concentrations of TNP-470 (10 nM) that are similar to therapeutic serum concentrations in animal models does not induce apoptosis (Yeh et al. 2000). Thus, it is unclear whether decreased eNOS Nα-myristoylation is relevant to the pharmacological effect of TNP-470 at therapeutic concentrations.

An analysis of total protein [3H]-myristic acid incorporation in BAECs after treatment with a lower cytostatic concentration of TNP-470 (50 nM) indicates that protein Nα-myristoylation is largely unaffected (Turk et al. 1999). Interestingly, the only exception was an unidentified 150-kDa protein whose signal decreases ~2-fold in intensity after TNP-470 treatment (Turk et al. 1999). Given that the MW of eNOS is ~150 kDa, these results suggest that Nα-myristoylation of eNOS may also be affected at lower cytostatic concentrations of TNP-470 (Turk et al. 1999). Thus, abnormally localized eNOS resulting from decreased Nα-myristoylation may still participate in the activation of the p53 pathway.

Another possibility is that Metinit retention may unexpectedly alter the turnover of a MetAP2-reliant protein (Griffith et al. 1997; Sin et al. 1997). For example, MetAP2 inhibition could directly activate the p53 pathway through the retention of Metinit on p53 or both p53 and p21WAF1/CIP1 protein, abnormally stabilizing these proteins. p53, however, retains Metinit under normal conditions and is unlikely to be directly affected by the inhibition of MetAP2 (Zhang et al. 2000). Although p21WAF1/CIP1 is a MetAP substrate (Met-Ser-), the requirement of p53 for p21WAF1/CIP1 expression also rules out

38 VETRO, DUMMITT, AND CHANG Chapter 2 a direct stabilization of p21WAF1/CIP1 by MetAP2 inhibition (Zhang et al. 2000).

5. CONCLUSION

Much work remains in understanding the functions of MetAP1 and MetAP2, their substrate specificities, and their subcellular distributions in higher animal cells. Further studies in these areas should help in elucidating the mechanism of lethality that results from Metinit retention as well as the role of MetAP2 catalytic activity in angiogenesis. It is hoped these insights will lead to new strategies for targeting angiogenesis in the treatment of vascular-related diseases.

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