Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins
© Marondedze et al.; licensee BioMed Central Ltd. 2013
Received: 10 May 2012
Accepted: 29 December 2012
Published: 5 January 2013
Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins.
Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus.
Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated.
† Enrichment of methionine oxidized peptides (oxMet) using TiO 2 with and without DHB
TiO2enrichment without DHB
TiO2enrichment with DHB
Assigned spectra oxMet pep.
In our proteomic analysis we considered a peptide as containing oxidised Met residue when it was identified with high confidence (≥ 95%) in at least two biological replicates. A total of 385 cGMP-dependent methionine oxidised proteins were identified (Additional file2, tab “AF1”). Assigned spectral counts (Additional file2, tab “AF2”) were used to estimate the relative ratio of peptides containing oxidized Met residue(s) as compared to total number of peptides identified in the sample.
An example of a tandem mass spectrometry result demonstrating oxidative modification of TiO2-enriched peptides extracted from 8-Br-cGMP-treated cells is shown in Figure2. Peptides containing single oxidised Met residue show an increase in mass to charge (m/z) ratio of 15.9994 that corresponds to the average mass of an oxygen atom. For example, the peptide fragment (DHDKPIQQVIAEMTDGGVDR) of the alcohol dehydrogenase 1 (AT1G77120) in non-oxidised form has the m/z ratio of 850.3723 (Figure2A), while after oxidation of Met residue, the m/z ratio shifts to 866.3673 (Figure2B).
Proteins enriched in the GO categories: 'response to oxidative stress', 'response to ROS', 'response to oxygen', and 'ROS metabolic process' showed four main patterns. (1) Loss of peptides or proteins containing oxidised Met residue(s) and reduction in the number of peptide copies after treatment, e.g. glyceraldehyde-3-phosphate dehydrogenase (AT1G13340; AT3G04120) and 60S acidic ribo-somal protein (AT2G27720). Loss of Met oxidised peptides can occur due to degradation of copies of proteins with oxidised Met residues or the reduction of oxidised Met residues. (2) Increase in the number of oxidised peptide fragments after treatment, e.g. peroxidase (AT2G22420; AT4G08770). This may imply that cGMP either indirectly induces over-expression of specific proteins with peptide fragments susceptible to oxidation or preferentially induces oxidation of Met residues in specific proteins without necessarily inducing transcription and/or translation. (3) New peptides detected with Met residues oxidised after treatment, e.g. ATP synthase (AT5G08670), and (4) multiple Met residues becoming oxidised after treatment, e.g. in the heat shock protein 70 (AT3G12580) (Additional File2, tab “AF1”).
cGMP-dependent proteins with multiple methionine residues oxidised and their enriched GO terms
*OxMet/total Met residues
Enriched GO term(s)
Low expression of osmotically responsive gene 1
RC, translational elongation
Male gametophyte defective 1
Co2+, Cu2+ and Zn2+ binding
GTP binding, GTP catabolic process, RCa
Heat shock protein 70
Responses to heat, bacterium, H2O2 and HLI, RCa, RTS, RV
Heat shock protein 70
Responses to heat, bacteria, H2O2, HLI, RCa, RTS, RV
Cytochrome C1 family
Heme binding, Fe2+ binding
GTP binding elongation factor Tu family protein
ATP, Co2+ and Zn2+ binding, RCa
Translocase outer membrane 20
Metal ion binding
Nascent polypeptide-associated complex
Transcription regulation and mitochondrial translocation
Highly methionine oxidised proteins after cGMP treatment
% of oxidised fragments
Enriched GO term(s)
Peroxidase superfamily protein
Oxidation-reduction process, response to oxidative stress
Response to salt stress, RC
Transcription regulation and mitochondrial translocation
Response to salt stress
Response to ABA stimulus, oxidative and salt stress, RCa
Heat shock protein 70
Defense response to bacteria and fungus, response to heat, and virus, RCa, RC
Cold, circadian rhythm and RNA binding 2
Regulation of stomatal movement, response to osmotic and salt stress, RCa, RC
Methylesterase PCR A
Metabolic process, negative regulation of catalytic activity
Cellular respiration, oxidation-reduction process, response to hypoxia, osmotic stress and salt stress, RCa
Mitochondrial malate dehydrogenase
Oxidation-reduction process, defense response to bacteria, response to salt stress, RCa, RC
Given that Met oxidation can profoundly alter cellular responses, the question is if there is evidence for site selectivity and if so, what determines it. In order to address this question, we have subjected all 385 proteins with one or several oxidised Met to further analysis and noted the following. Firstly, the average Met frequency in the set of proteins containing oxidised Met is 0.027 as compared to the other amino acids (AA) in the complete proteome where it is 0.025, and hence is nearly the same; we also noted that none of the 50 Arabidopsis proteins with the highest frequency of Met occurrence (0.09) contained any oxidised Met residues. This indicates that Met frequencies in proteins per se are not a factor that determines increased oxidation. Secondly, of the 385 proteins containing at least one modified Met residue, only eight residues were on the N-terminus, and of the 150 double Met (-MM-) residues, five proteins had both residues oxidised and eight proteins - only one. Thirdly, of the 575 oxidised Met residues detected, 75 had a glutamic acid (Glu) and 68 had an aspartic acid (Asp) as an immediate C-terminal neighbour and this bias is likely, at least in part, due to preferential enrichment of these AAs by TiO2, however we also find the uncharged alanine (Ala: 47) enriched on the C-terminus. The most frequent N-terminal neighbours are glutamic acid (Glu: 67) and again the uncharged alanine (Ala: 65). The least frequent C- and N-terminal neighbours are tryptophan (Trp: 2; 0, respectively) and cysteine (Cys: 1; 2, respectively). We further compared the observed frequency of amino acids flanking Met positions to their theoretically expected values implied from the analysis of overall AA frequencies in the entire Arabidopsis proteome. This process involved counting all of the occurrences of AAs in the proteome and establishing their relative frequency in the proteome, and the analysis was done in Matlab (Version R2010b). We note that, while under-represented (i.e. the observed frequency in positions flanking Met is lower than their average in the entire proteome), leucine and serine are the most frequent flanking AAs. In turn, Met flanking a Met is 35% over-represented on the N-terminal side and 25% over-represented on the C-terminal side. In contrast, oxidised Mets have much reduced relative preference for Met (-14% on N-terminus and -44% on C-terminus), albeit based on a very limited sample. Other under-represented flanking AAs are cysteine (Cys) and proline (Pro).
The rapidly evolving field of redox proteomics provides new evidence supporting the notion that oxidation of Met residues may have a great impact on protein activity, regulation of biochemical pathways and cellular function in response to changing environmental conditions. This is consistent with the observed Met oxidation accumulation in plants under low temperature conditions and the fact that plant methionine sulfoxide reductase (MSR) confers increased tolerance to freezing. It is also conceivable that the differential oxidation footprint of Met is a result of different susceptibility depending e.g. on the conformation of the protein or on differential access for repair of the MSR to different proteins or protein domains. Furthermore, our results are an indication that many of the Arabidopsis proteins involved in modulating the level of reactive oxygen species (ROS), including ROS-scavenging and ROS-producing proteins, may - at least in part - be regulated by oxidation of their Met residues.
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