MAPs and POEP of the roads from prokaryotic to eukaryotic kingdoms

Biochimie 82 (2000) 95−107 © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908400003837/REV

Review

MAPs and POEP of the roads from prokaryotic to eukaryotic kingdoms Bansidhar Datta* Department of Chemistry, University of Nebraska at Lincoln, Lincoln , NE 68588, USA (Received 3 October 1999; accepted 3 December 1999) Abstract — Methionine aminopeptidases (MAPs) play important roles in protein processing. MAPs from various organisms, for example E. coli, S. typhimurium, P. furiosus, Saccharomyces cerevisiae, and porcine have been purified to homogeneity and their MAP activities have been tested in vitro and in vivo. The DNA sequence analyses of MAP genes from the above organisms reveal sequence homologies with other prokaryotic MAPs as well as with various eukaryotic homologues of rat p67. The cellular glycoprotein, p67 protects the α-subunit of eukaryotic initiation factor 2 (eIF2) from phosphorylation by its kinases. We call this POEP (protection of eIF2α phosphorylation) activity of p67. The POEP activity of p67 is observed in different stress-related situations such as during heme-deficiency of reticulocytes, serum starvation and heat-shock of mammalian cells, vaccinia virus infection of mammalian cells, baculovirus infection of insect cells, mitosis, apoptosis, and possibly during normal cell growth. The POEP activity of p67 is regulated by an enzyme, called p67-deglycosylase (p67-DG). When active, p67-DG inactivates p67 by removing its carbohydrate moieties. Remarkable amino acid sequence similarities at the C-terminus of rat p67 with its eukaryotic and prokaryotic homologues which have MAP activities, raise several important questions: i) does rat p67 have MAP activity?; and ii) if it does have MAP activity, how the two activities (POEP and MAP) of p67 are used by mammalian cells during their growth and differentiation. In this review, discussions have been made to evaluate both POEP and MAP activities of p67 and their possible involvement during normal growth and cancerous growth of mammalian cells. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS methionine aminopeptidases / POEP activity / eukaryotic initiation factor / cellular glycoprotein p67

1. Introduction

2. Characteristics of methionine aminopeptidases from various organisms

Aminopeptidases are widely distributed in living organisms. These peptidases are classified according to their substrate specificities. One such class is the methionine aminopeptidases (MAPs) which remove N-terminal leading methionine from nascent peptides during the early stages of protein synthesis. The rate of protein synthesis in mammals is largely regulated by the phosphorylation of the α-subunit of eIF2. When phosphorylated at the α-subunit, eIF2 is inactive in the initiation of protein synthesis. A cellular glycoprotein, p67, keeps eIF2 in an active form by protecting its α-subunit from phosphorylation by eIF2-kinases (POEP activity). The eukaryotic homologues of rat p67 that include yeast, mouse, and human p67, have been cloned. The cDNA sequence analyses of p67 from the above species indicate that it may also have MAP activity. It is very important to understand the physiological significance of the two activities (POEP and MAP) of p67 during mammalian cell growth.

2.1. Biochemical characteristics of MAPs

* Correspondence and reprints

2.1.1. Mammals

The components (protein factors, ribosomes, and tRNAs) required for protein synthesis and its overall mechanistic steps (initiation, elongation, and termination) both in bacteria and mammals are remarkably conserved. In addition to several cofactors, the presence of other enzymes, for example aminopeptidases, is detected almost in all organisms including bacteria to human. Among several aminopeptidases, MAP drew more attention because of the unique role of methionine in the initiation of peptide chains, and its subsequent removal from the N-terminal of most nascent polypeptides by MAP during the early stages of peptide chain growth [1-4]. The existence of MAP activity in rabbit reticulocytes and in trout testis cells was detected as early as 1970 by Jackson and Hunter [1] and Wilson and Dintzis [2], and Wigle and Dixon [3], respectively. In 1971 Kerwar et al. [5] developed an assay to detect aminopeptidases using aminoacyl β-naphthylamide as a substrate and found that brain ribosomes from rabbit contain higher levels of MAP activity as compared to other aminopeptidases. However, this enzyme is loosely associated with the ribosomes since

96 the activity could be dissociated by washing the ribosomes with buffers of low ionic strength. In 1972 Yoshida and Lin [6] developed another assay where aminopeptidases could be detected by staining for peptidase activities in starch gels after electrophoresis of lysates of rabbit blood cells or various rabbit tissues. The MAP was identified on the basis of hydrolysis of the substrate Met-Gly-Met-Met into Met and Gly-Met-Met, and its specificity was determined by its inability to hydrolyze other tetrapeptides not starting with methionine. Released methionine was detected by an amino acid analyzer [6]. In the early 1980s, Freitas et al. [7] identified and later purified to homogeneity a rat MAP (RtMAP) which is membrane bound in mitochondria and microsomes. The substrate used to detect this enzyme is methionyl-lysyl-bradykinin (MetLys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). Interestingly however, this enzyme also has arylamidase activity [7]. Moreover, this enzyme can remove radiolabeledmethionine from nascent haemoglobin in rabbit reticulocytes [8]. The RtMAP has a molecular mass of 74 kDa. It is activated by beta-mercaptoethanol, and inhibited by p-hydroxymercuric benzoate indicating a SH-group dependent enzyme, and this dependence may account for its easy spontaneous inactivation. In addition, this enzyme is also inhibited by well known inhibitors of aminopeptidases, for example EDTA, puromycin, and bestatin, indicating that the RtMAP is distinct from several other metallo-aminopeptidases [9, 10]. Purification of porcine MAP (PcMAP) was achieved from either fresh or frozen liver. Enzyme prepared from frozen tissue generally was obtained in lower yield and contained a greater amount of a low molecular form. The PcMAP has a molecular mass of ≈ 70 kDa, it is monomeric in its native state, and its amino acid composition was relatively rich in glycine, alanine, and leucine [11]. Although this enzyme contains over 100 acidic plus amide residues which is more than twice the number of basic residues, the separation characteristics by Mono S chromatography indicated that the majority of the acidic residues are in the amidated form. Like MAPs from bacteria and yeast (see below), the activity of the PcMAP is stimulated by the biologically rarer metal ion Co2+, and inhibited by beta-meraptoethanol, EDTA, and relatively common metal ions, for example Mg2+, Mn2+, and Zn2+. The substrate specificity of PcMAP is very similar to bacterial MAP, but different from yeast MAP1 [11] (also see below). The PcMAP was digested with endopeptidase Lys-C, and pairs of sense and antisense degenerate oligonucleotides corresponding to two different peptides were used to pull out a 273-bp product that was later used to screen a porcine liver λgt10 cDNA library. A 2.1-kb clone was obtained and this DNA fragment was subsequently used to screen a cDNA library from human fetal brain. The cDNA sequence of the HmMAP was also reported independently by Li and Chang [13]. A ≈ 480 nucleotide long DNA probe was pulled out from a human cDNA

Datta library by performing a RT-PCR reaction where the primers used were designed from the reported cDNA sequence of the rat p67 [13]. Although the cDNA sequence corresponding to the 2.1-kb insert from porcine was not reported [11], that for the ≈ 2.4-kb cDNA insert from human was reported, and this sequence has > 92% sequence identity with that of rat cDNA [12, 13]. In contrast, the reported amino acid composition of PcMAP varies significantly from that of HmMAP obtained from the cloned cDNA sequence [11-13]. A dramatic difference in amino acid composition between HmMAP and PcMAP is observed where the numbers of alanine, glycine, and leucine residues in HmMAP are 37, 42, and 29 respectively as compared to 92, 82, and 98 respectively in PcMAP (table I). Although the combined numbers for acidic and amide residues are basically the same in both HmMAP and PcMAP, the number of basic residues differs in these two MAPs (for example the number of Arg plus Lys in HmMAP = 67 whereas that in PcMAP = 42) (table I). This indicates that the PcMAP may have overall more negative charge as compared to HmMAP. Other small but significant differences also exit in amino acid composition in MAPs from porcine, human, and rat p67 (compare the amino acids in bold in table I). The differences in amino acid composition between human and rat p67 are however very insignificant (table I). Taken together, these analyses indicate that the PcMAP gene is different from that of human and rat. It seems likely that mammals have two genes encoding MAPs, and PcMAP is encoded by the second gene. 2.1.2. Plants

The existence of MAPs from plants was deduced as early as 1993 in mitochondria from potato by amino acid sequence comparison of the N-terminus of purified cytochrome b with its predicted amino acid sequence from the cloned cDNA [14]. Its purification and cloning have not been reported yet. 2.1.3. Yeast

The existence of yeast MAP (yMAP) was reported as early as 1985 by Tsunasawa et al. [15], and determination of the specificity surrounding initiator methionine revealed that methionine is easily removed by yMAP when the penultimate residue is either Ala, Gly, Pro, Ser, Thr, and Val. In contrast, the activity of yMAP is inhibited when the penultimate residue is either Arg, Asn, Asp, Glu, Gln, Ile, Leu, Lys, and Met [15-17]. In general, small uncharged residues having radii of gyration < 1.29 Å promote removal of Met from the N-terminus whereas bulky hydrophobic and positively charged residues do not, indicating a major determinant for the selectivity of MAP. In addition to yMAP, these observations are also true for MAPs from other organisms as well [18, 19]. A major hurdle in purifying MAPs from eukaryotic organisms was their instability as well as contamination with non-specific

MAPs and POEP: from prokaryotic to eukaryotic kingdoms Table I. A comparison of amino acid composition of purified porcine MAP with that derived from cDNAs of rat p67 and its human homologue. The amino acid composition of the purified porcine MAP was determined experimentally [11], whereas those from either human or rat were determined from the reported cDNA sequences [12, 13, 84]. Amino acids A C D E F G H I K L M N P Q R S T V W Y

Species Human

Porcine*

Rat

37 15 39 42 10 42 11 26 43 29 9 17 23 12 25 22 27 27 3 15

92 N.D. 50 61 9 82 6 16 22 98 14 N.D. 27 – 20 37 39 40 N.D. 13

36 15 37 42 12 42 11 27 47 29 7 17 22 14 22 21 29 27 3 12

Significant differences in number of amino acids are shown in bold. N.D., not determined. The numbers of glutamine residues are not shown but they are included in the numbers of glutamic acid residues. *Data reproduced from reference [11].

aminopeptidases. In 1990 Chang et al. [20] purified a MAP (yMAP1) from aged yeast cells to homogeneity using Met-Ala-Ser as a substrate. Apparently, this yMAP1 is much more stable when yeast cells are kept at –20 °C for at least 4 months. The enzyme has a molecular mass of approximately 36 kDa that is close to the molecular masses of MAPs from E. coli and S. typhimurium (see below). The yMAP1 has an isoelectric point of 7.8, and it is inhibited by metalloprotease inhibitors (EDTA, o-phenanthroline and nitrilotriacetic acid) and sulfhydryl modifying reagents (HgCl2 and p-hydroxymercuribenzoic acid). However, this enzyme was not inhibited by pepstain, chymostatin, antipain, leupeptin, Trasylol, TLCK, or bestatin [20]. The yMAP1 is highly specific for methionine, and it follows a similar substrate specificity like those of E. coli and S. typhimurium ( [20] and see below). The yMAP1 also showed no detectable activity toward any of the representative amino acid p-nitroanilides (Ala, Arg, Glu, Gly, Leu, Met, Pro, or Val), indicating that the yMAP1 is different from all other yeast aminopeptidases [20]. Although the yMAP1 can be completely inhibited by 10 mM ZnCl2, it can also be activated by Co2+,

97 indicating that Co2+ may play an important role in the catalysis and stability of yMAP1 [20], as it does for the MAPs from E. coli or S. typhimurium. The yMAP1 was subjected to partial amino acid sequencing, and its cDNA was isolated from a yeast cDNA library using degenerated oligonucleotides. The yMAP1 contains two domains, a unique NH2-terminal region (≈ 120 amino acids), and a catalytic C-terminal domain which has amino acid sequence similarity with MAPs from E. coli (42.7%), S. typhimurium (43.7%), and B. subtilis (40.4%) [21]. At the N-terminus of yMAP1 there are two zinc-finger motifs which are essential for normal MAP function in vivo, even though in vitro enzyme assays indicate that they are not involved in catalysis [22]. Each of the COOH-terminal tails of the three ubiquitin precursors for example UBI1, UBI2, and UBI3 contain a zinc finger like motif. They associate with ribosomes and play a role in ribosomal biogenesis [23, 24]. Based on these observations, the yMAP1 has been presumed to associate with ribosomes, however, further proof is still lacking. Deletion of the yMAP1 gene results no lethality of yeast cells, although it does slow growth dramatically [21]. This observation led to the identification of another MAP (yMAP2) in yeast that rescued the slow growth phenotype. In addition, deletion of both yMAP1 and yMAP2 from yeast cells resulted in growth lethality [25]. Interestingly however, the yMAP2 protein sequence shows only 22% sequence identity over a region of 202 amino acids with yMAP1 [21, 25]. A marked difference between yMAP1 and yMAP2 is their N-terminal sequences. At the N-terminus, yMAP1 has two zinc fingers whereas yMAP2 has a lysine rich basic domain [21, 22, 25]. Both of these enzymes have five conserved residues that are also present in aminopeptidase P, prolidases, and creatinases, although these enzymes do not use Co2+ as a metal cofactor, but instead they use Mn2+ for their activity [26]. According to the three-dimensional structure of EcMAP (see below), the two cobalt atoms are coordinated by Asp97, Asp108, H171, Glu204, and Glu235 [27]. These amino acid residues are also present in eukaryotic MAPs [25], giving a domain consensus sequence for Co2+-binding D(X)10D(X)62-68H(X)32E(X)31-93E, where ‘X’ is any amino acid. The conserved residues of yMAP1 that are required for binding to Co2+, could also use the appropriate physiological metal ion Zn2+ for its MAP activity [28]. The alteration of one of the conserved residue for example Asp219 to Asn in yMAP1, resulted in a change of the requirement of the penultimate residue next to methionine. It has been suggested that Asp219 of yMAP1 might be important for the structural stabilization of the enzyme and that cobalt plays an important role in its structure. In addition, the yMAP1 (Asp219 to Asn) mutant can also interfere with the activity of yMAP2 in yeast cells, indicating that both yMAP1 and yMAP2 may have a common cellular target [29], or these enzymes are functioning in a same pathway. There seems to be another

98 yeast gene encoding protein (349 amino acids long) having ≈ 60% sequence similarity with rat p67, and disruption of its gene from yeast chromosome II resulted in no phenotypic effect in rich growth medium [30]. Based on the amino acid sequence similarity with rat p67 which is more related to yMAP2, it seems likely that the above yeast protein is related to yMAP2. 2.1.4. Bacteria

The activity of the bacterial MAP was detected as early as 1968 by Takeda and Webster [31], but its instability had prevented any significant purification [32]. One of the aminopeptidases purified from E. coli B showed higher levels of activity when bound to ribosomes as compared to the free enzyme [33]. In contrast, another enzyme, named aminopeptidase I, had methionine aminopeptidase activity and this enzyme was not associated with the ribosome. In addition, the substrate specificity of the later enzyme also suggests that it has some non-specific activity [32]. Significant progress had been made by Ben-Bassat et al. [34] who cloned the E. coli MAP gene (map) from the K-12 CM89 strain that is deficient in peptidases A, B, D, N, and Q by introducing a genomic DNA library that originated from the same strain of E. coli. The specific genomic clone that overproduced MAP ≈ 100-fold as compared to the parental strain, was characterized for further analysis. The substrate used for assaying EcMAP was Met-Gly-MetMet. A 1.2-kb fragment containing the E. coli map gene was sequenced. The open reading frame encodes 264 amino acids, and the calculated molecular mass is 29 333 as compared to the estimated size of 32 kDa for the EcMAP by SDS-PAGE. Although the N-terminal methionine is not present in purified EcMAP, its sequence matched with the predicted sequence from the cloned gene. The activity of EcMAP is inhibited by EDTA and stimulated by Co2+ which could not be substituted by Mn2+, Cu2+, Zn2+, or Mg2+. The activity of the EcMAP was severely reduced when Tris buffer was substituted for phosphate buffer, but the addition of sodium or potassium salts restored the MAP activity in vitro. The substrate specificity of this cloned MAP indicated that the penultimate residues with radii of gyration < 1.29 Å were selectively favored, although the removal of methionine from a substrate with Ile (radius of gyration is 1.56 Å) at the penultimate residue could not be explained by the above rule [34]. Overproduction of EcMAP in E. coli enhances the removal of the amino-terminal methionine from recombinant IL-2 and ricin A chain proteins in vivo [35]. Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-βthiogalactoside suggesting that EcMAP is essential for growth [36]. Using very similar approaches as described for the purification of EcMAP, Miller at al. purified MAP from S. typhimurium, determined its activity in vitro and in vivo,

Datta and also its specificity towards various tripeptides or recombinant IL-1β protein [37, 38]. The gene for S. typhimurium MAP (named pepM) has been cloned and it was found that it contained an ORF encoding 264 amino acid residues [39]. Further in vivo study indicated that the pepM gene indeed is absolutely required for the growth of the S. typhimurium [40]. Overexpression of the pepM can remove N-terminal methionine from IL-2 and granulocyte/macrophage colony-stimulating factor [41]. The substrate specificity and the metal requirement for MAP from E. coli and S. typhimurium are the same [34, 37]. The MAP from the hyperthermophilic archaeon, P. furiosus (PfMAP), has been cloned recently [42, 43]. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. Enzymatic activity and substrate specificity are very similar to other MAPs. The PfMAP also has a 62 amino acid insertion between the fourth and fifth cobalt binding domains [43]. Although it does not have the usual 90–165 amino acids extension that is present in eukaryotic MAPs, it is very homologous at the metal binding domains of eukaryotic MAPs as compared to bacterial MAPs. This indicates the possibility that the function of the metal binding domain of MAPs is phylogenetically conserved from bacteria to human, and this function is required for the survival of organisms such as E. coli, yeast, and possibly other organisms as well. Consistent with this is the finding that the bacterial MAP is required for the viability of E. coli [36] and S. typhimurium [40]. It is conceivable that during evolution, species have adapted by altering the structure of the MAP to better regulate its dominant function in order to control the growth of higher organisms. 2.2. Genetic and structural characteristics of MAPs from various organisms; their similarities and differences The cDNA sequences for MAPs of E. coli [34], S. typhimurium [39], P. furiosus [43], yeast [21, 25], Drosophila [44], and human [12, 13] have been reported. Based on the amino acid sequence analysis these are classified as type I (EcMAP and yMAP1) and type II (eukayotic MAPs including yMAP2 and PfMAP) [45-47]. Similarities between type I and type II MAPs include the conservation of the amino acid residues that are required for Co2+ binding and the overall geometry surrounding those amino acids [45, 46]. Detailed mutational analyses suggested the absolute requirement of all five cobalt binding amino acids for MAP activity of EcMAP [48]. Similar studies at the hydrophobic pocket that seems to be the substrate binding domain, revealed the requirements of both Cys70 and Trp221 of EcMAP for its MAP activity [48]. Intriguingly however, these two amino acids are not conserved in type II MAP. Within type II MAPs there are some species-specific variations. For example, the PfMAP has a 62 amino acid insertion whereas the HmMAP has a 64

MAPs and POEP: from prokaryotic to eukaryotic kingdoms amino acid insertion between the fourth and fifth cobalt binding amino acid residues [12, 13, 43]. The yMAP2 has a shorter N-terminal extension in which there is a basic lysine rich domain [25]. The HmMAP has two basic domains separated by an acidic residue-rich domain at its N-terminus [12, 13]. Based on several observations, it was clear that a major problem in identifying a MAP in cell extracts was the presence of several broad-specificity enzymes capable of hydrolyzing N-terminal methionine from peptides, and different MAP homologues in different species. One such example is that the RtMAP removes N-terminal methionine from oligopeptides and methionyl-2-naphthylamide but not from the substrate Met-Ala-Ser. The same substrate however has been used successfully to purify yMAP1 [10, 20]. In spite of a number of difficulties significant progress has been made to purify and characterize MAPs from various organisms. In order to get detailed insights into the substrate specificity of MAPs, it was to determine the structures of the MAPs from various important species. The first crystal structure of EcMAP at 2.4-Å resolution has been reported by Roderick and Matthews in 1993 [27]. The crystal structure of EcMAP shows remarkable pseudo two-fold symmetry with two cobalt ions at 4 Å below the midpoint and the active site is located at the junction of the two halves (figure 1). Although the apparent sequence and structural homology between the two halves of EcMAP are significant, the observed level of sequence identity among them is rather limited. These similar structural motifs are also found in two other classes of protease, namely, the chymotrypsinlike serine proteases [49] and the acid proteases [50]. An even more striking observation is the structural similarity between EcMAP and creatinase, although the former is a metallo-protein and the latter is not. This raises an important question: can homologous proteins evolve different enzymatic activities [51]? The three-dimensional structure of EcMAP also provides evidence for the existence of a single sterically accessible cavity for the putative substrate that is near the cobalt atoms. Electron density in this region however, does not suggest the presence of L-methionine, present in the crystal storage solution at 10 mM concentration. Adjacent to the metal binding site there is a hydrophobic pocket that seems to be the candidate for the binding of the amino-terminal methionine side chain or structure with peptide substrate (see below) required for substrate recognition and catalysis [27]. The crystal structure of HmMAP was reported recently [52]. Although the overall topology of MAPs, their active sites, and requirements of the metal ions for their activities from E. coli, P. furiousus, and human are the same, there are significant differences between HmMAP and EcMAP. One difference includes the presence of the 165 amino acid N-terminal extension and a 64 amino acid insertion between fourth and fifth cobalt

99 binding domains of HmMAP that breaks the pseudo two-fold symmetry of EcMAP. Another striking difference is the overall geometry of the hydrophobic pocket that had been assumed to be acting for substrate specificity and catalysis [52]. An anticaner drug, fumagillin which has been assumed as a substrate mimic with the epoxidebearing side chain resembling methionine’s side chain, can only bind HmMAP covalently at its His231 residue, and inhibits its MAP function [53-55]. From the structural superimposition of EcMAP with HmMAP it was predicted that His79 of EcMAP that is corresponding to His231 of HmMAP, is too far away from the hydrophobic pocket to form a bond with fumagillin. However, by using biophysical and biochemical methods such as mass spectrometry, electronic absorption, and N-terminal sequence analysis, Lowther et al. showed that His79 of EcMAP can bind to fumagillin [56]. It is however not known at this point whether the specificity pocket of the HmMAP would contain methionine when its crystal would have been grown or stored in solution containing L-methionine. Based on these observations, along with the finding for the strict requirement of the penultimate residue in order to cleave methionine from proteins, one can speculate that the overall geometry of methionine in combination with its penultimate residue may determine the catalytic activity of MAPs. If this is true, then one may find the presence of the methionine and its appropriate penultimate residue inside the specificity pocket of MAPs when crystals are grown in the presence of appropriate polypeptides. Indeed, detailed structural analysis of EcMAP at 1.9 Å resolution permitted visualization of the coordination geometry of EcMAP bound to a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-ValL-Phe-OMe at the metal binding domain [57]. It is also possible that more than one specificity pocket is present in MAPs and that some of them will open only in the presence of the appropriate substrate. Although significant progress has been made in determining the threedimensional structures of homologous proteins such as EcMAP and HmMAP, it is not clear where exactly the substrate binds to these proteins. It is worthwhile to note that there is a remarkable similarity among the chemical formulae of different substrate molecules of the MAP/ creatinase superfamily [51, 57]. 3. Approaches towards purifying recombinant human MAP and testing its activity The protein encoding open reading frame from human MAP cDNA was fused with gene encoding glutathionineS-transferase. The GST-fusion protein was purified from E. coli, and later used to test for MAP activity. In a typical MAP assay, the substrate Met-Gly-Met-Met was first labeled with a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, later

100

Datta

Figure 1. Overall polypeptide chain fold of EcMAP. The viewing direction is similar in both diagrams and is approximately parallel to the internal two-fold rotation axis. a. Schematic diagram of the ribbon structure. b. Stereoview of the α-carbon atoms, including the two active site cobalt ions shown as filled black circles. a and b are reproduced from Roderick and Matthews [27].

incubated with the enzyme (GST-MAP), and the products (Met and Gly-Met-Met) were separated by highperformance liquid chromatography [58]. Some MAP activity was detected in bacterially expressed human

recombinant HmMAP [59]. In contrast, the purified epitope-tagged HmMAP that was expressed in E. coli did not show any MAP activity (Wang, personal communication). Since MAP already is present in E. coli, and its

MAPs and POEP: from prokaryotic to eukaryotic kingdoms activity could be induced over 100-fold by artificially introducing a MAP gene [36], the expression of either the GST-fusion or his-tagged MAPs from other origins should not have any toxic effect in the E. coli cells. However, consistent reports from different laboratories indicate difficulties in obtaining purified active recombinant MAPs from E. coli. 4. Future questions regarding biochemical characteristics of MAPs from various organisms Significant progress has been made in identifying, purifying, characterizing, and cloning MAP genes from various organisms. Crystal structures of prokaryotic and eukaryotic MAPs are also determined. Still, satisfactory answers to several questions have not been obtained. The issue of ribosome binding of MAPs from various organisms remains unsolved, although the observation that the initiator methionine is removed from the nascent peptide of ≈ 20 amino acids long, makes it likely that MAPs should be ribosome bound. PfMAP is unusually stable at high temperature (≈ 100 °C). Analysis of the PfMAP structure in comparison with EcMAP and other mesophile proteins reveals several factors which may contribute to the hyperthermostability of PfMAP. These factors include a significantly high number of hydrogen bonds and ion-pairs between side-chains of oppositely charged residues involved in the stabilization of helices; an increased number of hydrogen bonds between the positively charged side-chains and neutral oxygens; a larger number of buried water molecules involved in cross-linking the backbone atoms of sequentially separate segments; and stabilization of two antiparallel β-strands connecting the two domains of the molecule by proline residues [60-62]. Overall, electrostatic interaction is an important factor stabilizing PfMAP. Based on several lists of biochemical evidence such as column chromatograpic separations, effects on various metal ions, effects on different pHs and other protease inhibitors, it is clear that prokaryotic or eukaryotic MAPs do not have similar hypothermic properties like PfMAP. Regardless of their origins, MAPs however have important functions in regulating protein stability, protein turnover, and thus protein levels [45, 47]. 5. Regulation of protein synthesis initiation in mammals Initiation of protein synthesis plays a central role in gene expression. A precise balance between proliferation and apoptosis of mammalian cells is regulated at the level of translation of the mature messages. Translation of the messages is largely regulated at the level of the formation of the initiation complex with eukaryotic initiation factor

101 2 (eIF2), Met-tRNAi, and 40S ribosome. This step plays a significant role in the initiation of peptide chain synthesis [63, 64]. However, this process is blocked when the smallest, α-subunit, of eIF2 is phosphorylated by its kinases. In various stress-related situations such as nutrient starvation, viral infection, heat-shock, and mitosis, the α-subunit of eIF2 is indeed phosphorylated in order to down-regulate the overall rate of protein synthesis [65]. This regulation of the rate of protein synthesis is highly conserved in eukaryotes [66]. The phosphorylation of the α-subunit of eIF2 is also regulated by the expression and activity of a cellular glycoprotein, p67 [64]. The discovery of p67 was reported in 1988 [67]. It is an eIF2-associated protein [67, 68], and eIF2 is bound to ribosomes [63, 64]. p67 protects the α-subunit of eIF2 from phosphorylation by kinases [67] (POEP activity). Subsequent studies revealed that p67 binds to wheat germ agglutinin (WGA) which recognizes terminal GlcNAc moieties [69] and even more specifically recognizes the -CONH2 group attached to the sugar [70]. Preincubation of hemin supplemented rabbit reticulocyte lysates with WGA inhibited protein synthesis. A monoclonal antibody against p67 was characterized from mice by immunizing the animal with a protein complex containing eIF2 and p67. Preincubation of hemin supplemented rabbit reticulocyte lysates with this monoclonal antibody against p67 also inhibited in vitro protein synthesis. In both cases the inhibition of protein synthesis was accompanied by the increased phosphorylation of the α-subunit of eIF2 [71]. Treatment of p67 whether in a free form or eIF2-bound, with hexasominidase for 36 h, or with sodiumborohydrate in the presence of mild alkali, prevents WGA or the monoclonal antibody against p67 to recognize p67 protein. However a polyclonal antibody against p67 detects the backbone of the treated protein [69]. In addition, isolated or eIF2-bound p67 when incubated with galactosyltransferase in the presence of 3H-labeled UDPgalactose was radio-labeled as detected by fluorography [69]. Taken together these in vitro studies led to the suggestion that p67 contains O-GlcNAc moieties, and these glycosyl residues are required for p67 POEP activity. Several in vivo studies also suggested that the increase rate of protein synthesis in cultured mammalian cells correlated with high levels of p67 and reduced levels of phosphorylation of the α-subunit of eIF2. On the other hand, a decreased rate of protein synthesis in mammalian cells also correlated with low levels of p67 and increased levels of phosphorylation of the α-subunit of eIF2. These in vivo conditions include proliferation of mammalian cells [72], mitosis [73], viral infections (namely vaccinia virus [74] and baculovirus infections [75]), heat shock [76], and programmed cell death [77]. There is also a good correlation between deglycosylation of p67 and its subsequent inactivation during mitosis [73]. The plant homologue of rabbit p67, named PKI, has been identified, and it has been shown to bind plant

102 double-stranded RNA activated protein kinase, PKR. The PKI protein was maximally detectable during seed germination and its level declined rapidly to a non-detectable level soon after leaf emergence. The PKI level is again detectable at the mid-milk stage in seed development [78]. During these transitions of seed germination there is a good correlation between high levels of eIF2α phosphorylation and low levels of PKI. These results suggested the involvement of PKI in plant development, and the mechanism of p67 action is very similar in plants as compared to other organisms, for example rat, mouse, rabbit, and human. The cloning of a cDNA for PKI has not been reported yet. 6. Positive and negative regulations of p67 activity to protect the α-subunit of eIF2 When mammalian cells in culture were serum starved, the expression of p67 was inhibited significantly. Expression and POEP activity of p67 are however induced during mitogenic stimulation [79-81]. Similarly, the expression and POEP activity of p67 are upregulated at the later stages of heat shock of mammalian cells [76]. These inductions of the levels of the message and p67 protein are very reminiscent of the expression of the immediate early genes, for example c-fos and c-jun, during mitogenic stimulation [82]. Indeed, the cloning and detailed analysis of the p67 promoter from rat revealed the presence of TRE and AP1 sites and these cis-acting elements are essential for the induced expression of p67 in response to mitogens [83]. Likewise, the heat-shock element (HSE) at the rat p67 promoter also is required for its induced expression during the later stages of heat-shock of mammalian cells [76]. Another important regulation of the p67 activity is at the level of post-translational modification, namely glycosylation. The presence of the terminal O-GlcNAc moieties that could be recognized by WGA, are required for POEP activity of p67. At the later stages of viral infections [74, 75], heme deficiency in reticulocytes [71], and during mitosis [73], there is an upregulation of the level and activity of an enzyme called p67-deglycosylase (p67-DG) which inactivates p67 by removal of the terminal GlcNAc moieties. This inactivation of p67 leads to the increased levels of phosphorylation of the α-subunit of eIF2 (figure 2). 7. Cloning of a cDNA corresponding to rat p67 In 1993 Wu et al., reported the cloning of a cDNA for rat p67 [84]. Based on the amino acid sequences corresponding to peptides that were obtained by digesting purified rabbit p67 with either trypsin or cyanogen bromide, degenerate oligonucleotides were synthesized and used as primers to perform a RT-PCR reaction in total

Datta

Figure 2. A schematic representation of the POEP activity of p67. When the α-subunit of eIF2 is phosphorylated by its kinases, it becomes inactive in initiation of protein synthesis in eukaryotes. The cellular glycoprotein, p67, inhibits this process by protecting eIF2α from phosphorylation. The glycosyl (GlcNAc) moieties attached to p67 are required for its activity. p67-DG, when active, removes the sugar moieties from p67 and inactivates it. This leads to the increased phosphorylation of eIF2α and thus inhibition of protein synthesis.

cytoplasmic RNA isolated from rat hepatoma tumor cells (KRC-7). A ≈ 329 bp DNA fragment was obtained and this DNA fragment was later used as a probe to screen a cDNA library constructed in λgt11 using mRNAs from cultured KRC-7 cells as templates. A 2.0-kb insert from the cloned phage was later subcloned in various expression vectors and the gene product was characterized by various biochemical approaches. The cloned cDNA encodes a 480 amino acid protein with a molecular mass of 53 kDa that was predicted for the unglycosylated protein. In the cloned cDNA for rat p67 there are several unique features which include the presence of two lysine rich basic domains separated by an acid rich domain at the N-terminus of the protein (figure 3). In the C-terminus of rat p67, five cobalt binding amino acids that are conserved in MAPs from various organisms, are also present [84]. Recent cloning reports from various MAPs indicated a strong homology especially at the C-terminal 166–480 amino acids of rat p67 with other MAPs [43, 60], raising an important question whether rat p67 has MAP activity. 8. Does rat p67 have MAP activity? Our first attempt to test for the MAP activity of the cloned rat cDNA for p67 was unsuccessful. In this attempt we made a GST-fusion chimeria with rat p67 that was purified from E. coli cells. Although most of the GSTfusion protein was degraded, whatever was left in the sample did not show any detectable MAP activity (unpublished). The degradation of GST-p67 (rat) could not be prevented by using excess protease inhibitors during the purification steps. To overcome this problem of degradation we later purified his-tagged rat p67 from E. coli, and it was sent to Dr. J. Wang at Abbott laboratories for testing its MAP activity. Although the degradation problem was partially solved, apparently this his-tagged rat p67 purified from E. coli did not show any detectable MAP or POEP

MAPs and POEP: from prokaryotic to eukaryotic kingdoms

Figure 3. A schematic representation of the amino acid sequences of eukaryotic homologues of rat p67. The number of amino acids (#AAs) and percentage identities in amino acid sequences of p67 among various species are shown. The thin vertical bars at the beginning of the solid bars represent two lysine-rich domains separated by an acidic residue-rich domain.

activity either (unpublished). Several factors which include the absence of the rarer metal ion (Co2+) during the purification of the fusion proteins or the lack of proper post-translational modifications such as glycosylation in E. coli, may contribute to this lack of MAP or POEP activity of the recombinant p67. It is also possible that the lack of glycosylation of p67 in E. coli could lead to the mis-folded recombinant p67 that is inactive in both MAP and POEP activity. In another study, an epitope-tagged human p67 was expressed and purified from E. coli. Unfortunately, this recombinant protein also did not show any detectable MAP activity (J. Wang, personal communication). Given the significant sequence similarity at the C-terminus of rat p67 with MAPs from other organisms makes it quite likely that this part of the molecule may have MAP activity. On the other hand, the unique N-terminus of p67 is involved in its POEP activity. Indeed, our latest finding suggests that the N-terminal 159 amino acids are sufficient for the POEP activity of p67 (to be published elsewhere). Taken together these results raise the question of how two such activities of p67 are used by mammalian cells. An intriguing possibility could be that the MAP activity of p67 is inhibitory to its POEP activity or vice versa. Consistent with this hypothesis is the absence of the unique N-terminus of p67 in lower organisms where the growth of cells is not regulated at the level of phosphorylation of eIF2 or its analogs. During evolution, the rate of protein synthesis is indeed highly regulated in higher eukaryotes. Detailed mutational analyses of rat p67 and their effects on p67 activities in vivo and in vitro are in progress in my laboratory to answer the several questions raised above. 9. Does rat p67 bind to ribosome? To test for ribosome binding of rat p67, an indirect approach was taken. The entire coding region of rat p67

103 was fused to the gene encoding green fluorescence protein (GFP). The fusion chimera was constitutively expressed in rat KRC-7 cells. Examination of the transfected cells under the microscope did not show any green signal from the nucleolus - the site for ribosome biogenesis. However, pronounced green signals were detected mostly in the cytoplasm including the Golgi apparatus - the site for post-translational modifications such as glycosylation (to be published elsewhere). These intriguing results indicate that rat p67 may not be tightly associated with the ribosome, but that it carries out its post-translational modification in the Golgi. It will be interesting and challenging to test both in vivo and in vitro whether other eukaryotic homologues of rat p67 (figure 3) also exhibit POEP activity. Indeed, some in vivo correlations exist where homologues of rat p67 play roles in the regulation protein translation. The differential accumulation of transcript of Drosophila homologue of rat p67 throughout embryonic and larval development agrees closely with the accumulation of two other regulators of translation initiation in Drosophila: the cap-binding protein eIF4E and the eIF2α kinase DGCN2 [44]. Likewise, the plant homologue of rat p67 also exhibits the role as a translational regulator during seed germination [78]. 10. Role(s) of p67-DG in the regulation of p67 activity The unusual post-translational modification, such as addition of O-linked GlcNAc moieties on p67, may be a major determinant in regulating its activity. The presence of an enzyme, p67-DG, in rabbit reticulocytes has been detected and reported as early as 1993 [71]. This enzyme when active, removes the glycosyl residues from p67, making it inactive in POEP activity. During stress-related situations such as virus infections, especially vaccinia virus infection of mammalian cells and baculovirus infection in insect cells, there was an up-regulation of p67-DG activity [74, 75]. This increased activity of p67-DG deactivated p67 and subsequently increased phosphorylation of the α-subunit of eIF2. This may explain in part the possible mechanism of the shut-off of host-protein synthesis during early stages of vaccinia virus infection of mammalian cells. In addition to its stabilizing activity of p67, hemin either keeps p67-DG in an inactive form or prevents the production of p67-DG at the transcriptional level or at the post-translational level [75]. Saha et al. [75] purified the p67-DG from baculovirus infected insect cells to homogeneity, and the protein was later microsequenced. The sequences of at least six peptide fragments matched the sequence of the endochitinase precursor which is one of the baculovirus core proteins (unpublished). Interestingly however, the polyclonal antibody generated against the protein with p67-DG activity crossreacted with a cellular protein [75] which could be the viral homologue of the mammalian endochitinase. Endo-

104

Figure 4. A hypothetical model for the regulation of cell growth by p67 and p67-DG. During normal cellular growth (A), there is a balance between the cellular concentrations of p67 and p67-DG. When this balance is perturbed by more of the p67 protein (B) or more of the p67-DG protein (C), cells may grow abnormally or die with apoptosis respectively.

chitinases remove GlcNAc moieties from glycoproteins [85]. Recently, the purification of p67-DG from rabbit reticulocytes has been accomplished [73]. From combined immunological and biochemical studies, its ability to remove GlcNAc moieties from p67 in vitro and in vivo, has been established [73]. The cloning of the p67-DG cDNA is underway in my laboratory. The sequence of the cloned p67-DG cDNA will definitely clarify whether the mammalian endochitinase is the same as purified p67-DG. Cellular functions of p67 and p67-DG indicate that the appropriate ratio of the effective cellular concentrations of p67 and p67-DG will possibly determine the fate of the cells whether they will grow or die with apoptosis (figure 4). Unregulated differentiation or apoptosis of mammalian cells may cause neoplasia. The latest findings about the binding of p67 with the anticancer drug fumagillin or its derivatives raise the question of the possible involvement of p67 during angiogenesis, as discussed below. Angiogenesis is the process of forming new blood vessels from existing ones either by sprouting or by splitting from their vessels of origin. The physiological importance of the angiogenesis is to regulate the female reproductive cycle, and to repair, remodel and regenerate tissues during wound healing. The pathological importance of angiogenesis is due to its central role in diabetic retinopathy, rheumatoid arthritis, psoriasis, atherosclerotic plaques, and cancer [86]. Several angiogenic stimulators and inhibitors have been characterized. Reports from several laboratories suggest that the ratio between stimulators and inhibitors precisely balances angiogenesis or

Datta apoptosis during neovascularization [87, 88]. Almost all of the angiogenic inhibitors known today also inhibit proliferation of endothelial cells, and these are found in a fragment of a larger protein which itself lacks inhibitory activity [89]. An exception to this are the small molecule angiogenic inhibitors that are isolated from fungi and bacteria [90, 91]. Many of the protein angiogenic inhibitors are indeed generated by tumors, and released to the blood stream that subsequently inhibit endothelial proliferation and metastasis [92, 93]. A surprising correlation exists where the inhibition of p67 function(s) from endothelial cells with anticancer drugs, fumagillin or its derivatives, resulted in the inhibition of cell growth [52-55, 94]. Mammalian cells (from rat and human) with low levels of p67 undergo apoptosis because of the inhibition of the rate of global protein synthesis [77]. Angiogenesis needs active translational machinery to synthesize several cytoskeleton proteins that are required to form new blood vessels. Further studies are needed to understand the molecular mechanisms of the regulation of the p67 activities in order to understand the molecular basis of the angiogenesis and its therapeutic control during pathological conditions.

Acknowledgments In this review I have tried hard to bring all the wonderful work done in this field by excellent laboratories. I would also like to apologize to other investigators whose important contributions I have missed unintentionally and were not included here. I would like to thank Dr. J. Wang from Abbott Laboratories for testing his-tagged rat p67 for its MAP activity, and letting me cite his unpublished observation. I am very grateful to Professor John W.B. Hershey (University of California, Davis, CA, USA) for his excellent comments and suggestions during preparation of the manuscript. This work was supported by the National Institute of Health/National Center for Research Resources Grant GM59190.

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