Specificity of cotranslational amino-terminal processing of proteins in yeast

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.     

The C. elegans methionine aminopeptidase 2 analog map-2 is required for germ cell proliferation

We have investigated the physiological function of type 2 methionine aminopeptidases (MetAP2) using Caenorhabditis elegans as a model system. A homolog of human MetAP2 was found in the C. elegans genome, which we termed MAP-2. MAP-2 protein displayed methionine aminopeptidase activity and was sensitive to inhibition by fumagillin. Downregulation of map-2 expression by RNAi led to sterility, resulting from a defect in germ cell proliferation. These observations suggest that MAP-2 is essential for germ cell development in C. elegans and that this ubiquitous enzyme may play important roles in a tissue specific manner.     

Treatment of cells with the angiogenic inhibitor fumagillin results in increased stability of eukaryotic initiation factor 2-associated glycoprotein, p67, and reduced phosphorylation of extracellular signal-regulated kinases

Fumagillin, an angiogenic inhibitor, binds to methionine aminopeptidase 2, which is the same as eukaryotic initiation factor 2-associated glycoprotein, p67. p67 protects eIF2alpha from phosphorylation by its kinases. To understand the importance of fumagillin binding to p67, we measured the level of p67 in mouse C2C12 myoblasts treated with fumagillin. We show that fumagillin increases the stability of p67 by decreasing its turnover rate. The increased levels of p67 result in inhibition of phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERKs 1 and 2). p67 binds to these ERKs, and the 108-480 amino acid segment is sufficient for this binding. p67's affinity to ERKs 1 and 2 also increases in fumagillin-treated myoblasts while its affinity for eIF2alpha remains unchanged. A mutant at the conserved amino acid residue D251A increases the phosphorylation of ERKs 1 and 2 without affecting the binding to p67, thus indicating the importance of this residue in the regulation of the phosphorylation of these ERKs. These results suggest that fumagillin increases the stability of p67 and its affinity to ERKs 1 and 2 and causes the inhibition of the phosphorylation of ERKs 1 and 2.     

Molecular cloning,expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC6803

Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 (map-1), slr0918 (map-2) and sll0555 (map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.Abbreviations map Gene encoding methionine aminopeptidase - MetAP Methionine aminopeptidase - eMetAP-Ia Escherichia coli methionine aminopeptidase type Ia - yMetAP-Ib Yeast methionine aminopeptidase type Ib - yMetAP-IIa Yeast methionine aminopeptidase type IIa - hMetAP-IIb Human methionine aminopeptidase type IIb - pfMetAP–IIa Pyrococcus furiosis methionine aminopeptidase type Ia - bst MetAP-Ia Bacillus stearothermophilus methionine aminopeptidase type Ia - c1MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-1 - c2MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-2 - c3MetAP-Ib Cyanobacterial methionine aminopeptidase type Ib, ncoded by map-3     

The function of N alpha-acetylation of the eye-lens crystallins

The putative protective role of the N alpha-acetyl group of proteins has been investigated. Synthetic, non-acetylated N-terminal tetrapeptides of the alpha A2- and gamma II-crystallin chains are good substrates for leucine aminopeptidase, while the acetylated ones are completely resistant. In the native, non-acetylated, gamma-crystallin the N terminus is not degraded by leucine aminopeptidase. Newly synthesized alpha A2-crystallin, in which the normally occurring N-terminal acetylation has been prevented during cell-free translation, is virtually resistant against degradation by leucine aminopeptidase. Only at extreme enzyme-substrate ratios the N-terminal methionine is removed. Although the N alpha-acetyl group by its very nature protects against this exopeptidase, we conclude that the group is not essential for this purpose in the native crystallins.     

Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase

Amino-terminal processing in the yeast Saccharomyces cerevisiae has been investigated by examining numerous mutationally altered forms of iso-1-cytochrome c. Amino-terminal residues of methionine were retained in sequences having penultimate residues of arginine, asparagine, glutamine, isoleucine, leucine, lysine, and methionine; in contrast, the amino-terminal methionine residues were exercised from residues of alanine, glycine, and threonine and were partially excised from residues of valine. The results suggest the occurrence of a yeast aminopeptidase that removes amino-terminal residues of methionine when they precede certain amino acids. A systematic search of the literature for amino-terminal sequences formed at initiation sites suggests the hypothetical yeast aminopeptidase usually has the same specificity as the amino peptidase from bacteria and higher eukaryotes. Our results and the results from the literature search suggest that the aminopeptidase cleaves amino-terminal methionine when it precedes residues of alanine, glycine, proline, serine, threonine, and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glutamine glutamic acid, isoleucine, leucine, lysine, or methionine. In contrast to the normal iso-1-cytochrome c and in contrast to the majority of the mutationally altered proteins, certain forms were acetylated including the following sequences: acetyl(Ac)-Met-Ile-Arg-, Ac-Met-Ile-Lys, Ac-Met-Met-Asn-, and Ac-Met-Asn-Asn-. We suggest yeast contains acetyltransferases that acetylates these mutant forms of iso-1-cytochromes c because their amino-terminal regions resemble the amino-terminal regions of natural occurring proteins which are normally acetylated. The lack of acetylation of closely related sequences suggest that the hypothetical acetyltransferases are specific for certain amino-terminal sequences and that the 3 amino-terminal residues may play a critical role in determining these specificities.     

Processing of the N termini of nascent polypeptide chains requires deformylation prior to methionine removal.

N-formyl-methionine termini are formed in the initiation reaction of bacterial protein synthesis and processed during elongation of the nascent polypeptide chain. We report that the formyl group must be removed before the methionine residue can be cleaved by methionine aminopeptidase. This has long been implicitly assumed, but that assumption was based on inconclusive data and was in apparent conflict with more recently published data. We demonstrate that the Salmonella typhimurium methionine aminopeptidase is totally inactive on an N-formyl-methionyl peptide in vitro, and present a detailed characterization of the substrate specificity of this key enzyme by use of a very sensitive and quantitative assay. Finally, a reporter protein expressed in a strain lacking peptide deformylase was shown to retain the formyl group confirming the physiological role of the deformylase.     

Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).     

Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.     

Microsomal methionine aminopeptidase: properties of the detergent-solubilized enzyme

A methionine aminopeptidase (MAP) found in rat liver microsomes behaves as membrane-bound enzyme. Triton-solubilized MAP when chromatographed on DEAE-cellulose columns was separated from other microsomal arylamidases. The enzyme hydrolyzes N-terminal methionine from methionyl-lysyl-bradykinin (Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) being then characterized as a typical aminopeptidase. It also shows preferential arylamidase activity upon Met-2-naphthylamide. MAP was activated by 2-mercaptoethanol and inhibited by p-hydroxymercuribenzoate. Contrarily to other well characterized aminopeptidases, MAP was not affected by EDTA, puromycin or bestatin. Altogether these data suggest that MAP is a unique microsomal enzyme distinct from other previously described aminopeptidases. It could be involved in the removal of methionine from nascent peptides during protein synthesis.     

Insights into the mechanism of Escherichia coli methionine aminopeptidase from the structural analysis of reaction products and phosphorus-based transition-state analogues.

In an effort to differentiate between alternative mechanistic schemes that have been postulated for Escherichia coli methionine aminopeptidase (eMetAP), the modes of binding of a series of products and phosphorus-based transition-state analogues were determined by X-ray crystallography. Methionine phosphonate, norleucine phosphonate, and methionine phosphinate bind with the N-terminal group interacting with Co2 and with the respective phosphorus oxygens binding between the metals, interacting in a bifurcated manner with Co1 and His178 and hydrogen bonded to His79. In contrast, the reaction product methionine and its analogue trifluoromethionine lose interactions with Co1 and His79. The interactions with the transition-state analogues are, in general, very similar to those seen previously for the complex of the enzyme with a bestatin-based inhibitor. The mode of interaction of His79 is, however, different. In the case of the bestatin-based inhibitor, His79 interacts with atoms in the peptide bond between the P(1)' and P(2)' residues. In the present transition-state analogues, however, the histidine moves 1.2 A toward the metal center and hydrogen bonds with the atom that corresponds to the nitrogen of the scissile peptide bond (i.e., between the P(1) and P(1)' residues). These observations tend to support one of the mechanistic schemes for eMetAP considered before, although with a revision in the role played by His79. The results also suggest parallels between the mechanism of action of methionine aminopeptidase and other "pita-bread" enzymes including aminopeptidase P and creatinase.     

Characterization of N alpha-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences' hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.     

Genetic and biochemical definition of the bovine papillomavirus E5 transforming protein.

Mutations surrounding the first methionine codon of the E5 transforming gene of bovine papillomavirus (type 1) were analyzed for their effect on cellular transformation and on the synthesis of the 7-kd E5 polypeptide. Frameshift mutations upstream of this methionine codon (bp 3879) affect neither transforming activity nor the ability to synthesize full-size E5 protein. In contrast, frameshift mutations distal to this position result in the inhibition of cell transformation and prevent synthesis or accumulation of E5 protein in cells containing the mutant viral genomes. Several in-frame mutations distal to the first methionine codon have a minimal effect on transforming activity but alter the electrophoretic mobility of the E5 protein in a manner consistent with the generated genetic alteration (deletion, insertion or substitution). In all cases where the protein is detected, it fractionates with cellular membranes and forms dimers. These studies indicate that (i) the methionine codon at bp 3879 serves as the initiation codon for the mature E5 protein, (ii) changing the charge of the E5 amino-terminus (from neutral to positive) does not prevent the association of this hydrophobic polypeptide with cellular membranes, and (iii) E5 amino-terminal mutations do not interfere with the ability of this polypeptide to form homodimers. We conclude that the major focus-inducing activity of the intact BPV genome is due to the function of the small polypeptide encoded in the 3' half of the E5 ORF.     

Bacterial aminopeptidases: Properties and functions

Abstract: Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues from polypeptides and proteins. Bacteria display several aminopeptidasec activities which may be localised in the cytoplasm, on membranes, associated with the cell envelope or secreted into the extracellular media. Studies on the bacterial aminopeptide system have been carried out over the past three decades and are significant in fundamental and biotechnological domains. At present, about one hundred bacterial aminopeptidases have been purified and biochemically studied. About forty genes encoding aminopeptidases have also been cloned and characterised. Recently, the three-dimensional structure of two aminopeptidases, the methionine aminopeptidase from Escherichia coli and the leucine aminopeptidase from Aeromonas proteolytica , have been elucidated by crystallographic studies. Most of the quoted studies demonstrate that bacterial aminopeptidases generally show Michaelis-Menten kinetics and can be placed into either of two categories based on their substrate specificity: broad or narrow. These enzymes can also be classified by another criterium based on their catalytic mechanism: metallo-, cysteine- and serine-aminopeptidases, the former type being predominant in bacteria. Aminopeptidases play a role in several important physiological processes. It is noteworthy that some of them take part in the catabolism of exogenously supplied peptides and are necessary for the final steps of protein turnover. In addition, they are involved in some specific functions, such as the cleavage of N-terminal methionine from newly synthesised peptide chains (methionine aminopeptidases), the stabilisation of multicopy ColE1 based plasmids (aminopeptidase A) and the pyroglutamyl aminopeptidase (Pcp) present in many bacteria and responsible for the cleavage of the N-terminal pyroglutamate.     

An investigation of arterial insufficiency in rat hindlimb. An enzymic, mitochondrial and histological study.

A membrane-bound aminopeptidase able to remove methionine from haemoglobin nascent peptides is described. The enzyme also hydrolyses methionine from methionyl-lysyl-bradykinin but not lysine from lysyl-bradykinin. The tripeptide Met-Ala-Ser is poorly hydrolysed. This aminopeptidase also splits amino acid 2-naphthylamides, being, however, less specific with respect to these synthetic substrates.     

Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2.

The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression). One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes. We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus. These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores. These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.     

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.     

Synthesis and evaluation of 2-pyridyl pyrimidines with in vitro antiplasmodial and antileishmanial activity

A series of 2-pyridyl pyrimidines, reported inhibitors of Plasmodium falciparum methionine aminopeptidase 1b were synthesized and evaluated for their antiplasmodial activities. An analysis of physicochemical properties demonstrated a link between lipophilicity and antiparasitic activity. Cross screening of the library against cultured Leishmania donovani parasites revealed this class of compounds as potent inhibitors of parasite development in vitro.