N-terminal Methionine Cleavage Research Articles

Structural insights into N-terminal methionine cleavage by the human mitochondrial methionine aminopeptidase, MetAP1D

Isozymes are enzymes that catalyze identical biological reactions, yet exhibit slight variations in structures and catalytic efficiency, which enables the precise adjustment of metabolism to fulfill the specific requirements of a particular tissue or stage of development. Methionine aminopeptidase (MetAP) isozymes function a critical role in cleaving N-terminal methionine from nascent proteins to generate functional proteins. In humans, two distinct MetAP types I and II have been identified, with type I further categorized into cytosolic (MetAP1) and mitochondrial (MetAP1D) variants. However, despite extensive structural studies on both bacterial and human cytosolic MetAPs, the structural information remains unavailable for human mitochondrial MetAP. This study was aimed to elucidate the high-resolution structures of human mitochondrial MetAP1D in its apo-, cobalt-, and methionine-bound states. Through a comprehensive analysis of the determined structures and a docking simulation model with mitochondrial substrate peptides, we present mechanistic insights into the cleavage process of the initiator methionine from mitochondrial proteins. Notably, despite the shared features at the active site between the cytosolic and mitochondrial MetAP type I isozymes, we identified distinct structural disparities within the active-site pocket primarily contributed by two specific loops that could play a role in accommodating specific substrates. These structural insights offer a basis for the further exploration of MetAP isozymes as critical players in cellular processes and potential therapeutic applications.

Whole-cell modeling of E. coli confirms that in vitro tRNA aminoacylation measurements are insufficient to support cell growth and predicts a positive feedback mechanism regulating arginine biosynthesis.

In Escherichia coli, inconsistencies between in vitro tRNA aminoacylation measurements and in vivo protein synthesis demands were postulated almost 40years ago, but have proven difficult to confirm. Whole-cell modeling can test whether a cell behaves in a physiologically correct manner when parameterized with in vitro measurements by providing a holistic representation of cellular processes in vivo. Here, a mechanistic model of tRNA aminoacylation, codon-based polypeptide elongation, and N-terminal methionine cleavage was incorporated into a developing whole-cell model of E. coli. Subsequent analysis confirmed the insufficiency of aminoacyl-tRNA synthetase kinetic measurements for cellular proteome maintenance, and estimated aminoacyl-tRNA synthetase kcats that were on average 7.6-fold higher. Simulating cell growth with perturbed kcats demonstrated the global impact of these in vitro measurements on cellular phenotypes. For example, an insufficient kcat for HisRS caused protein synthesis to be less robust to the natural variability in aminoacyl-tRNA synthetase expression in single cells. More surprisingly, insufficient ArgRS activity led to catastrophic impacts on arginine biosynthesis due to underexpressed N-acetylglutamate synthase, where translation depends on repeated CGG codons. Overall, the expanded E. coli model deepens understanding of how translation operates in an in vivo context.

Investigating the Conditionally Essential Role of Human METAP1

CRISPR‐based forward genetic screens make it possible to systematically identify genes required for survival and growth in human cells, and therefore provide a powerful approach to characterize protein function and uncover targetable vulnerabilities in human cancer cells. Notably, it has become appreciated that gene essentiality can be context‐dependent, as CRISPR‐based loss‐of‐function screens across hundreds of human cancer cell lines have uncovered cell‐essential genes that can vary with cell‐intrinsic characteristics such as genotype and lineage. Importantly, environmental factors also influence cell physiology. However, there has been little consideration into how nutrient availability might impact gene essentiality, and most CRISPR‐based screens of human cells are performed in vitro using culture media that poorly recapitulate nutrient conditions in the body. To test the hypothesis that medium composition influences gene essentiality, our lab recently performed genome‐wide CRISPR screens of human blood cancer cell lines in traditional versus Human Plasma‐Like Medium (HPLM), a physiologic medium that we previously developed to more closely reflect the metabolic composition of human blood. Among the strongest HPLM‐essential hits identified from our screen results is METAP1, which encodes one of two human enzymes (METAP1 and METAP2) that can catalyze the cleavage of N‐terminal methionine from nascent polypeptides. Interestingly, these two METAPs are co‐expressed across most human cell lines, are each localized to the cytosol, and also share seemingly similar substrate preferences. However, our screen results indicate that endogenous METAP2 cannot complement the loss of METAP1in HPLM‐cultured cells, suggesting that METAP1 serves a non‐redundant and context‐dependent cell‐essential role. Therefore, we hypothesize that identifying the gene‐nutrient interaction that underlies conditional METAP1essentiality will provide key insights into this unforeseen role. Importantly, we have engineered METAP1‐knockout cells and, in turn, confirmed the conditional CRISPR phenotype for METAP1by performing relative growth assays. Therefore, we are now poised to identify the gene‐nutrient interaction and will proceed to use tools in biochemistry, metabolomics, and molecular biology to explore the context‐dependent role of METAP1 in proliferating human cells.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

A Proteogenomic Survey of the Medicago truncatula Genome

Peptide sequencing by computational assignment of tandem mass spectra to a database of putative protein sequences provides an independent approach to confirming or refuting protein predictions based on large-scale DNA and RNA sequencing efforts. This use of mass spectrometrically-derived sequence data for testing and refining predicted gene models has been termed proteogenomics. We report herein the application of proteogenomic methodology to a database of 10.9 million tandem mass spectra collected over a period of two years from proteolytically generated peptides isolated from the model legume Medicago truncatula. These spectra were searched against a database of predicted M. truncatula protein sequences generated from public databases, in silico gene model predictions, and a whole-genome six-frame translation. This search identified 78,647 distinct peptide sequences, and a comparison with the publicly available proteome from the recently published M. truncatula genome supported translation of 9,843 existing gene models and identified 1,568 novel peptides suggesting corrections or additions to the current annotations. Each supporting and novel peptide was independently validated using mRNA-derived deep sequencing coverage and an overall correlation of 93% between the two data types was observed. We have additionally highlighted examples of several aspects of structural annotation for which tandem MS provides unique evidence not easily obtainable through typical DNA or RNA sequencing. Proteogenomic analysis is a valuable and unique source of information for the structural annotation of genomes and should be included in such efforts to ensure that the genome models used by biologists mirror as accurately as possible what is present in the cell.

Experimental annotation of post-translational features and translated coding regions in the pathogen Salmonella Typhimurium

BackgroundComplete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. However, determining protein-coding genes for most new genomes is almost completely performed by inference using computational predictions with significant documented error rates (> 15%). Furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function.ResultsWe experimentally annotated the bacterial pathogen Salmonella Typhimurium 14028, using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. The data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in Salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. Additionally, we uncovered 12 non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel ORFs in Salmonella pathogenesis. We also characterized post-translational features in the Salmonella genome, including chemical modifications and proteolytic cleavages. We find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. Our in vivo proteolysis data identified more than 130 signal peptide and N-terminal methionine cleavage events critical for protein function.ConclusionThis work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of Salmonella as a resource for systems analysis.

ChemInform Abstract: Further Studies on the Chemical Cleavage of an N-Terminal Extra Methionine from Recombinant Methionylated Proteins.

An additional methionine residue of recombinant human growth hormone (hGH) was converted into an oxoacyl form with glyoxylic acid, copper(II) sulfate and pyridine, and then cleaved from the rest of the protein with 3,4-diaminobenzoic acid in the presence of 1 M AcOH and 2 M HCO2Na. The conditions for N-terminal methionine cleavage worked better than the previous conditions with 1,2-phenylenediamine. The same protocol was also applicable to the methionylated forms of recombinant human betacellulin (BTC), neurotrophin-3 (NT-3) and human interleukin-2 (IL-2). The conversion yield for hGH, BTC, NT-3 and IL-2 increased up to approximately 80, 70, 55 and 50%, respectively. These results indicate that non-methionylated recombinant proteins could be prepared from the methionylated derivatives by chemical methods.

Amino Acid Sequence Determination of Protein Biomarkers of Campylobacter upsaliensis and C. helveticus by “Composite” Sequence Proteomic Analysis

We have identified the protein biomarkers observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five strains of Campylobacter upsaliensis and one strain of C. helveticus by "bottom-up" proteomic techniques. Only one C. upsaliensis strain had previously been genomically sequenced. The significant findings are as follows: (1) The protein biomarkers identified were: 10 kD chaperonin, protein of unknown function (DUF465), phnA protein, probable periplasmic protein, D-methionine-binding lipoprotein MetQ, cytochrome c family protein, DNA-binding protein HU, thioredoxin, asparigenase family protein, helix-turn-helix domain protein, as well as several ribosomal and conserved hypothetical proteins. (2) Amino acid substitutions in protein biomarkers across species and strains account for variations in biomarker ion mass-to-charge (m/z). (3) The most common post-translational modifications (PTMs) identified were cleavage of N-terminal methionine and N-terminal signal peptides. The rule that predicts N-terminal methionine cleavage, based on the penultimate residue, does not appear to apply to C. upsaliensis proteins when the penultimate residue is threonine. (4) It was discovered that some protein biomarker genes of the genomically sequenced C. upsaliensis strain were found to have nucleotide sequences with GTG or TTG "start" codons that were not the actual start codon (ATG) of the protein based on proteomic analysis. (5) Proteomic identification of the protein biomarkers of the non-genomically sequenced C. upsaliensis and C. helveticus strains involved identification of homologous protein amino acid sequences to that of the sequenced strain. Interestingly, some protein sequence regions that were not completely homologous to the sequenced strain, due to amino acid substitutions, were found to have homologous sequence regions from more phyogenetically distant species/strains, e.g., C. jejuni. Exploiting this partial homology of more distant species/strains, it was possible to construct a "composite" amino acid sequence using multiple non-overlapping sequence regions from both phylogenetically proximate and distant strains. The new composite sequence was confirmed by both MS and MS/MS data. Thus, it was possible in some cases to determine the amino acid sequence of an unknown protein biomarker from a genomically non-sequenced bacterial strain without the necessity of either genetically sequencing the biomarker gene or resorting to de novo MS/MS analysis of the full protein sequence.

Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: Human serum albumin as a case study

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.

The Specificity in Vivo of Two Distinct Methionine Aminopeptidases in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential function of amino-terminal methionine removal is provided cotranslationally by two methionine aminopeptidases (MetAP1 and MetAP2). To examine the individual processing efficiency of each MetAP in vivo, we measured the degree of N-terminal methionine cleavage from a series of mutated glutathione-S-transferase (GST) proteins isolated from yeast wild-type, a map1 deletion strain, a map2 deletion strain, and a map1 deletion strain overexpressing the MAP2 gene. We found that MetAP1 plays the major role in N-terminal methionine removal in yeast. Both MetAPs were less efficient when the second residue was Val, and MetAP2 was less efficient than MetAP1 when the second residue was Gly, Cys, or Thr. These findings indicate that MetAP1 and MetAP2 exhibit different cleavage efficiencies against the same substrates in vivo. Interestingly, although methionine is considered a stabilizing N-terminal residue, we found that retention of the initiator methionine on the Met-Ala-GST mutant protein drastically reduced its half-life in vivo.

Further studies on the chemical cleavage of an N-terminal extra methionine from recombinant methionylated proteins

An additional methionine residue of recombinant human growth hormone (hGH) was converted into an oxoacyl form with glyoxylic acid, copper(II) sulfate and pyridine, and then cleaved from the rest of the protein with 3,4-diaminobenzoic acid in the presence of 1 M AcOH and 2 M HCO2Na. The conditions for N-terminal methionine cleavage worked better than the previous conditions with 1,2-phenylenediamine. The same protocol was also applicable to the methionylated forms of recombinant human betacellulin (BTC), neurotrophin-3 (NT-3) and human interleukin-2 (IL-2). The conversion yield for hGH, BTC, NT-3 and IL-2 increased up to approximately 80, 70, 55 and 50%, respectively. These results indicate that non-methionylated recombinant proteins could be prepared from the methionylated derivatives by chemical methods.

Bacterial aminopeptidases: properties and functions.

Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues from polypeptides and proteins. Bacteria display several aminopeptidasic 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.

Biological and immunological importance of Bet v 1 isoforms.

In 2D-PAGE analysis of Bet v 1, the major birch pollen allergen, up to 12 isoforms can be demonstrated that differ in their isoelectric points from about pH 4.9 to pH 5.9. The molecular weights of these isoforms seem to be rather similar, but minor variations can also be seen. Preliminary experiments with birch leaves seem to indicate that in aging leaves some isoforms can be found that do not occur in pollen. In birch cells cultured in vitro, Bet v 1 isoforms can be induced by bacterial infection that do not occur in pollen (Swoboda et al. (1995), Pant, Cell and Environment 18, 865-874). In a recent paper (Swoboda et al (1995)., J. Biol. Chem. 270, 2607-2613) we show that in natural Bet v 1 from pollen the isoforms are due to different protein sequences. The derived protein sequences of 10 different isoforms (corresponding to 13 different cDNAs) were determined and confirmed by plasma desorption mass spectrometry of purified natural Bet v 1 after trypsin and endoproteinase Glu-C digestion. These experiments also showed that pollen Bet v 1 isoforms were reactive to patients' sera to different degrees and that common post-synthetic modifications (besides N-terminal methionine cleavage) did not occur on Bet v 1. Recombinant isoforms were produced in E. coli, purified and tested with selected patients allergic to birch pollen (Ferreira et al., J. Exp. Med., in the press). The pattern of IgE binding to Bet v 1 isoforms widely differs. Also, T-cell clones from individual patients in some cases are specific to peptides occurring only in certain isoforms. It was of particular interest that three of the naturally occurring pollen Bet v 1 isoforms do not or hardly bind IgE of untreated patients allergic to Bet v 1. However, a comparison of IgE reactivity in patients before and after conventional immunotherapy with natural pollen extract clearly showed that this form of immunotherapy induced IgE to the isoforms that had been unreactive in untreated patients. One of these, Bet v 1d, showed a particularly strong potency towards T-cell stimulation. The isoform(s) that do not bind IgE in untreated patients but still show T-cell reactivity could be potentially utilized for a new form of immunotherapy that avoids the risk of anaphylaxis.

N-terminal variants of fatty acid-binding protein from bovine heart overexpressed in Escherichia coli

An expression vector for bovine heart fatty acid-binding protein (H-FABP) was constructed by introducing the coding part of the cDNA into the pET-3d vector. Transformed Escherichia coli strain BL21 (DE3)pLysS produced functional recombinant H-FABP up to 40% of the soluble proteins. The expression of fatty acid-binding protein was under the control of the T7-phi 10 promoter and the corresponding T7-RNA-polymerase in turn was induced by isopropyl β- d-thiogalactopyranoside. By combination of cation exchange chromatography and gel filtration pure recombinant protein was obtained exhibiting isoelectric heterogeneity. Recombinant H-FABP was resolved into at least six variants with isoelectric points between 5.1 and 5.6. After separation by preparative isoelectric focusing the four major variants were digested with trypsin and the resulting peptides were characterized by high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) mass spectrometry, amino acid sequencing and chemical modification. The structural differences were traced back to the N-termini beginning with either methionine, as expected from the cDNA, or methionine sulfoxide, valine and N-formyl methionine. The latter three arise from oxidation, cleavage of N-terminal methionine and incomplete deformylation, respectively.

Purification and Properties of an Aminopeptidase from Escherichia coli

Abstract An aminopeptidase from Escherichia coli has been purified to homogeneity and crystallized. This peptidase is responsible for about 65% of the hydrolytic activity found in crude extracts toward the substrate methionylalanylserine. It resembles the so-called leucine aminopeptidase of hog kidney in several respects, including size, metal requirements, and peptide specificity. Evidence based on substrate specificity suggests that this E. coli peptidase is not involved in cleavage of N-terminal methionine from nascent protein chains.