N-terminal protein processing: a comparative proteogenomic analysis

Abstract

N-terminal methionine excision (NME) and N-terminal acetylation (NTA) are two of the most common protein post-translational modifications. NME is a universally conserved activity and a highly specific mechanism across all life forms. NTA is very common in eukaryotes but occurs rarely in prokaryotes. By analyzing data sets from yeast, mammals and bacteria (including 112 million spectra from 57 bacterial species), the largest comparative proteogenomics study to date, it is shown that previous assumptions/perceptions about the specificity and purposes of NME are not entirely correct. Although NME, through the universal enzymatic specificity of the methionine aminopeptidases, results in the removal of the initiator Met in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val, the comparative genomic analyses suggest that this specificity may vary modestly in some organisms. In addition, the functional role of NME may be primarily to expose Ala and Ser rather than all seven of these residues. Although any of this group provide "stabilizing" N termini in the N-end rule, and de facto leave the remaining 13 amino acid types that are classed as "destabilizing" (in higher eukaryotes) protected by the initiator Met, the conservation of NME-substrate proteins through evolution suggests that the other five are not crucially important for proteins with these residues in the second position. They are apparently merely inconsequential players (their function is not affected by NME) that become exposed because their side chains are smaller or comparable to those of Ala and Ser. The importance of exposing mainly two amino acids at the N terminus, i.e. Ala and Ser, is unclear but may be related to NTA or other post-translational modifications. In this regard, these analyses also reveal that NTA is more prevalent in some prokaryotes than previously appreciated.

Fig. 1.

Two alternative cases for NME function. A, NME exposes seven residues to be new N termini of proteins. Because this is presumably for some functional reason, the conventional assumption is that all seven residues must have functional importance as N termini. By the comparative genomics postulate (as defined in the text), evolutionary conservation of all seven at P2 should be observed. If all of these residues are not conserved, one of the two assumptions must be incorrect; either not all seven residues are important or the comparative genomics postulate is invalid. B, Given that the comparative genomics postulate holds, and only two of the seven residues are of functional importance as N termini, then the other five residues are inconsequential players and only these two residues should be evolutionarily conserved.

Fig. 2.

Sequence motifs for the first ten residues of each protein determined by MS/MS. A, 6811 samples which retained Met and B, 9700 samples where Met was cleaved.

Fig. 3.

Comparison of MetAP specificities in Brachybacterium faecium, E. coli, and Deinococcus radiodurans. B. faecium, E. coli, and D. radiodurans significantly differ in NME specificity (Met occurring before Asn was released in 90% of cases in B. faecium). The specificity of D. radiodurans differs from expected with very few Thr residues exposed (only 10 of 82 samples).

Fig. 4.

Results from PTM searches in Saccharomyces cerevisiae. A, Breakdown of NME, non-NME, and NME+NTA (NME and Nα-acetylation) events. Approximately 65% of N-terminal events in yeast were found to be Nα-acetylations. B, Residue breakdown of the P2 position of proteins that underwent NME+NTA. Ser is strongly favored in yeast, much as Ser-conservation is also pronounced in yeast. C, 221 identifications of only NME+NTA proteins show that Ser dominates the composition. D, The intersection between NME+NTA and NME-only show a wide distribution among NME residues.

Fig. 5.

Distribution of identifications in 45 different bacterial organisms. Any proteins appearing in both sets are only counted once. Residues with greater than or equal to 1% representation are labeled in each pie chart. A, Aggregation of Venn diagrams of N-terminal PTM runs on 68 million bacterial spectra across 45 organisms. Of the 2613 NME+NTA, 8933 NME, and 6613 non-NME identifications, the only considerable overlap is across NME+NTA and NME, which is expected. A Venn diagram for each organism was constructed, and the counts for each set and overlapping sets were aggregated into this larger diagram. Sequelog relationships were not taken into account in the diagrams creation. 16% of bacterial N-terminal events were found to be Nα-acetylations. B, 8933 NME events with Ser, Ala, and Thr well represented. C, 2613 Nα-acetylation identifications (NME+NTA) where Ser, Ala, and Thr are again the major residues. D, The intersection between NME+NTA and NME yields 1216 identifications, comprising mostly of Ser and Thr.

Fig. 6.

Offset counts for the range of −150 to +150 in A. variabilis. NME events are represented as peaks located at −131, NME and Nα-acetylation at −89. Retention of Met (no NME event) is represented by a peak at 0.

Fig. 7.

Mean NME-conserved protein counts are shown for P2 through P5 for bacterial, yeast, and mammalian data sets. An NME-conserved protein is one containing the residue at the position in question (two through five) in X across all sequelogs of that protein. All values are obtained from bootstrap estimated mean and standard deviation of each statistic. A The set of residues considered is defined as X = {G,A,S,P,V,T,C}. It is evident that P2 contains a larger number of NME-conserved than P3-P5. B NME-conserved protein counts, without Ala and Ser, are shown for P2 through P5 for the three data sets. Here the X set does not contain Ala and Ser, and is defined as X = {G,P,V,T,C}. Not including Ala and Ser in X produces no elevated counts at P2 compared with P3, P4, and P5. This suggests that the boost in NME-conserved counts seen in A can be attributed to the Ala and Ser residues.

Fig. 8.

Conservation of residues are shown for P2 through P5 for data sets from A, Shewanella B, Saccharomyces C, mammalian. Ala and Ser are by far the most conserved in P2 across all three data sets. The number of proteins with Ala in the second position in Shewanella, Saccharomyces, and the mammalian data sets are 79, 24, and 541, respectively. The number of proteins with Ser in P2 in Shewanella, Saccharomyces, and mammalian data sets are 75, 130, and 268, respectively.