The effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M-catalyzed hydrolysis of enkephalins.

High performance liquid chromatography and high performance liquid chromatography/electrospray ionization-mass spectrometry were used to study the effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M (EC 3.4.11.2)-catalyzed hydrolysis of methionine (Met-enk) and leucine enkephalins (Leu-enk). Acetylation imparts a significant enhancement in the proteolytic stability of these two peptides. After 30 min of the reaction, < 10% of both acetylated enkephalins was hydrolyzed. In an 8-h incubation period, only a maximum of 54% acetylated (Ac)-Met-enk and 38% Ac-Leu-enk was hydrolyzed. Vmax and Km [infil] for the degradation of Ac-Met-enk were 1.4 nmol/min/50 ng and 2.2 mM, respectively. The corresponding values for the reaction of Ac-Leu-enk were 0.5 nmol/min/50 ng and 0.9 mM. Also, the aminopeptidase M activity on Met-enk can be inhibited in the presence of Ac-Met-enk, which acts as a mixed-type inhibitor with the inhibition constant (K(i)) of I x 10(-3) M.     

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.     

Identification of methionine Nalpha-acetyltransferase from Saccharomyces cerevisiae

N alpha-Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and was believed until now to be catalyzed by a single N alpha-acetyltransferase. The transfer of an acetyl group from acetyl coenzyme A to the alpha-amino group of five NH2-terminal residues (serine, alanine, methionine, glycine, and threonine) in proteins accounts for approximately 95% of acetylated residues. We have found that a crude lysate from Saccharomyces cerevisiae mutant (aaa1) deficient in N alpha-acetyltransferase activity can effectively transfer an acetyl group to peptides containing NH2-terminal methionine but not to serine or alanine. This methionine N alpha-acetyltransferase has been extensively purified, and this purified enzyme can selectively transfer an acetyl group to various model peptides containing an NH2-terminal methionine residue and a penultimate aspartyl, asparaginyl, or glutamyl residue. Such specificity of N alpha-acetylation of methionine has been previously observed based on the analysis of eukaryotic protein sequences (Persson, B., Flinta, C., Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527; Arfin, S.M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984). The indentification of this methionine N alpha-acetyltransferase provides an explanation as to why two distinct classes of N alpha-acetylated proteins exist in nature: (i) those whose initiator methionine is acetylated and (ii) those whose penultimate residue is acetylated after cleavage of the initiator methionine.     

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.     

Discriminating between Lysine Sumoylation and Lysine Acetylation Using mRMR Feature Selection and Analysis

Post-translational modifications (PTMs) are crucial steps in protein synthesis and are important factors contributing to protein diversity. PTMs play important roles in the regulation of gene expression, protein stability and metabolism. Lysine residues in protein sequences have been found to be targeted for both types of PTMs: sumoylations and acetylations; however, each PTM has a different cellular role. As experimental approaches are often laborious and time consuming, it is challenging to distinguish the two types of PTMs on lysine residues using computational methods. In this study, we developed a method to discriminate between sumoylated lysine residues and acetylated residues. The method incorporated several features: PSSM conservation scores, amino acid factors, secondary structures, solvent accessibilities and disorder scores. By using the mRMR (Maximum Relevance Minimum Redundancy) method and the IFS (Incremental Feature Selection) method, an optimal feature set was selected from all of the incorporated features, with which the classifier achieved 92.14% accuracy with an MCC value of 0.7322. Analysis of the optimal feature set revealed some differences between acetylation and sumoylation. The results from our study also supported the previous finding that there exist different consensus motifs for the two types of PTMs. The results could suggest possible dominant factors governing the acetylation and sumoylation of lysine residues, shedding some light on the modification dynamics and molecular mechanisms of the two types of PTMs, and provide guidelines for experimental validations.     

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.     

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.     

Identification of N-terminal acetylation of recombinant human prothymosin α in Escherichia coli

Functional modification of protein through N-terminal acetylation is common in eukaryotes but rare in prokaryotes. Prothymosin α is an essential protein in immune stimulation and apoptosis regulation. The protein is N-terminal acetylated in eukaryotes, but similar modification has never been found in recombinant protein produced in prokaryotes. In this study, two mass components of recombinant human prothymosin α expressed in Escherichia coli were identified and separated by RP-HPLC. Mass spectrometry of the two components showed that one of them had a 42 Da mass increment as compared with the theoretical mass of human prothymosin α, which suggested a modification of acetylation. The mass of another one was equal to that of the theoretical one. Peptides mass spectrometry of the modified component showed that the 42-Da mass increment occurred in the N-terminal peptide domain, and MS/MS peptide sequencing of the N-terminal peptide found that the acetylated modification occurred at the N-terminal serine residue. So, part of the recombinant human prothymosin α produced by E. coli was N-terminal acetylated. This finding adds a new clue for the mechanism of acetylated modification in prokaryotes, and also suggested a new method for production of N-terminal modificated prothymosin α and thymosin α1.     

N-Terminal Acetylation of Two Major Latex Proteins from Arabidopsis thaliana Using Electrospray Ionization Tandem Mass Spectrometry

The primary structure of two proteins named major latex protein in Arabidopsis thaliana were characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometer and Nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) after two-dimensional gel electrophoresis separation. We revealed that the two proteins with the same N termini and the N-terminal alanine were acetylated after methionine cleavage by fragmentation of three doubly charged peptides using a quadrupole-time of flight 2 tandem mass spectrometer. It was worth noting that one peptide with sodium addition and acetylation was sequenced. It is usually difficult to analyze the peptide sequence of sodium adduct due to the 22-Da increment. The two proteins are highly homologous, and both their N-terminal and C-terminal peptides were sequenced. Of the two proteins, gi|15236568 (spot A) appears only in the seeding stage and flower organ, but gi|15236566 (spot B) appears throughout the whole life of A. thaliana. The biological mechanism of the two proteins and the function of N-terminal acetylation remain to be elucidated. This study showed that ESI-MS/MS was a powerful tool for the characterization of N-terminal acetylation of proteins.     

Selective removal of N-terminal methionine from recombinant human growth hormone by an aminopeptidase isolated from Aspergillus flavus

An aminopeptidase was isolated from a soil fungus, which specifically cleaves the unnatural N-terminal methionine in recombinant human growth hormone. Reaction mixtures with different ratios of aminopeptidase to recombinant methionyl human growth hormone showed that the removal of N-terminal methionine was complete at 1:200 (w/w), and more than 90% complete at ratios up to 1:2000 (w/w) when incubated for 24 h at 37°C. The data indicate that the aminopeptidase we have purified can be used for the efficient conversion of unnatural recombinant proteins to their natural form. Received 18 September 1997/ Accepted in revised form 17 April 1998     

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b₅ at the N-terminal of interferon (b₅-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b₅ at the C-terminal of interferon (Met-IFN-b₅-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b₅-chimera), resolved this problem and gave enhanced biological activity.     

Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae

Methionine aminopeptidase (MAP), which catalyzes the removal of NH2-terminal methionine from proteins, was isolated from Saccharomyces cerevisiae. The enzyme was purified 472-fold to apparent homogeneity. The Mr of the native enzyme was estimated to be 36,000 +/- 5,000 by gel filtration chromatography, and the Mr of the denatured protein was estimated to be 34,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 7.0, and its pI is 7.8 as determined by chromatofocusing on Mono P. The enzyme was inactivated by metalloprotease inhibitors (EDTA, o-phenanthroline and nitrilotriacetic acid), sulfhydryl-modifying reagents (HgCl2 and p-hydroxymercuribenzoic acid), and Zn2+. Yeast MAP failed to cleave methionine p-nitroanilide. Among 11 Xaa-Ala-Ser analogues (Xaa = Ala, Asp, Gln, Glu, Ile, Leu, Lys, Met, Phe, Pro, and Ser), MAP cleaved only Met-Ala-Ser. MAP also cleaved methionine from other tripeptides whose penultimate amino acid residue is relatively small and/or uncharged (e.g. Pro, Gly, Val, Thr, or Ser) but not when bulky and/or charged (Arg. His, Leu, Met, or Tyr). Yeast MAP displayed similar substrate specificities compared with those of Escherichia coli (Ben-Bassat, A., Bauer, K., Chang, S.Y., Myambo, K., Boosman, A., and Chang, S. (1987) J. Bacteriol. 169, 751-757) and Salmonella typhimurium MAP (Miller, C., Strauch, K. L., Kukral, A. M., Miller, J. L., Wingfield, P. T., Mazzei, G. J., Werlen, R. C., Garber, P., and Movva, N. R. (1987) Proc. Natl, Acad. Sci. U.S.A. 84, 2718-2722). In general, the in vitro specificity of yeast MAP is consistent with the specificity observed in previous in vivo studies in yeast (reviewed in Arfin, S. M., and Bradshaw, R. A. (1988) Biochemistry 27, 7979-7984).     

N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins

N(alpha)-terminal acetylation occurs in the yeast Saccharomyces cerevisiae by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively. The N-terminal sequences required for N-terminal acetylation, i.e. the NatA, NatB, and NatC substrates, were evaluated by considering over 450 yeast proteins previously examined in numerous studies, and were compared to the N-terminal sequences of more than 300 acetylated mammalian proteins. In addition, acetylated sequences of eukaryotic proteins were compared to the N termini of 810 eubacterial and 175 archaeal proteins, which are rarely acetylated. Protein orthologs of Ard1p, Nat3p and Mak3p were identified with the eukaryotic genomes of the sequences of model organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus and Homo sapiens. Those and other putative acetyltransferases were assigned by phylogenetic analysis to the following six protein families: Ard1p; Nat3p; Mak3p; CAM; BAA; and Nat5p. The first three families correspond to the catalytic subunits of three major yeast NATs; these orthologous proteins were identified in eukaryotes, but not in prokaryotes; the CAM family include mammalian orthologs of the recently described Camello1 and Camello2 proteins whose substrates are unknown; the BAA family comprise bacterial and archaeal putative acetyltransferases whose biochemical activity have not been characterized; and the new Nat5p family assignment was on the basis of putative yeast NAT, Nat5p (YOR253W). Overall patterns of N-terminal acetylated proteins and the orthologous genes possibly encoding NATs suggest that yeast and higher eukaryotes have the same systems for N-terminal acetylation.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

地中海富盐菌PhaE蛋白乙酰化修饰对其功能的影响

【目的】研究地中海富盐菌PHA合酶(Pha EC)中Pha E亚基乙酰化修饰对其功能的影响,探讨乙酰化修饰对菌体生理代谢的调控作用。【方法】蔗糖密度梯度离心收集PHA颗粒,质谱鉴定颗粒结合蛋白Pha E的乙酰化位点。将乙酰化位点(赖氨酸,K)分别突变为精氨酸(R)(模拟去乙酰化)或谷氨酰胺(Q)(模拟乙酰化),利用同源双交换原理,将突变后的基因原位敲入基因组。以野生型为对照,检测突变对菌体生长、葡萄糖消耗和PHA合成能力的影响。利用Western blot检测PHA颗粒上Pha E的含量,进一步分析乙酰化修饰对蛋白功能的影响。【结果】在Pha E蛋白105位和170位赖氨酸(K)2个位点检测到乙酰化修饰。利用遗传操作系统将突变的基因原位敲入,共得到6种突变株。发酵结果表明,任何一种单突变对菌体生长及PHA合成的影响均不明显。但当2个位点同时突变成精氨酸(K105R/K170R)时,突变株生长及合成PHA的能力均受到明显抑制,2个位点同时突变成谷氨酰胺(K105Q/K170Q)则无明显影响。进一步的Western blot结果表明,突变成精氨酸的双突变株的PHA颗粒上,Pha E蛋白的含量相较于野生型约降低了一半。【结论】Pha E蛋白的去乙酰化能够导致菌株利用葡萄糖合成PHA的能力显著降低,其可能原因是降低了Pha E与PHA颗粒或PHA颗粒上Pha C的结合能力,从而降低了Pha EC合酶的活性。     

Transfer-messenger RNA-SmpB Protein Regulates Ribonuclease R Turnover by Promoting Binding of HslUV and Lon Proteases

RNase R, an important exoribonuclease involved in degradation of structured RNA, is subject to a novel mechanism of regulation. The enzyme is extremely unstable in rapidly growing cells but becomes stabilized under conditions of stress, such as stationary phase or cold shock. RNase R instability results from acetylation which promotes binding of tmRNA-SmpB, two trans-translation factors, to its C-terminal region. Here, we examine how binding of tmRNA-SmpB leads to proteolysis of RNase R. We show that RNase R degradation is due to two proteases, HslUV and Lon. In their absence, RNase R is stable. We also show, using an in vitro system that accurately replicates the in vivo process, that tmRNA-SmpB is not essential, but it stimulates binding of the protease to the N-terminal region of RNase R and that it does so by a direct interaction between the protease and SmpB which stabilizes protease binding. Thus, a sequence of events, initiated by acetylation of a single Lys residue, results in proteolysis of RNase R in exponential phase cells. RNase R in stationary phase or in cold-shocked cells is not acetylated, and thereby remains stable. Such a regulatory mechanism, dependent on protein acetylation, has not been observed previously in bacterial cells.