On 2017 May 16, Michael Tatham commented:

Is CoAlation biologically relevant or a non-functional by-product of the chemical reaction between CoA and cysteine thiols in proximal proteins under certain redox conditions?

Firstly, in my opinion the work described here is technically sound. The development of the specific antibody for CoA, and the mass spectrometric method to detect the modification on peptides are key tools in the analysis of any post-translational modification. However, there is a risk when using these super-sensitive methods, that one can detect vanishingly small amounts of modified peptides, which inevitably calls relevance into question. More specifically, modern mass-spectrometry based proteomics in combination with peptide-level enrichment of modified species has allowed us to identify modification sites in the order of tens of thousands for phosphorylation, ubiquitination, acetylation and SUMOylation (as of May 2017). For these fields, the onus of the researcher has very quickly shifted from identification of sites, to evidence for biological meaning. In short, the question is no longer “Which proteins?”, but “Why?”.

Taking acetylation as an example: Phosphositeplus (www.phosphosite.org) lists over 37000 acetylation sites, the majority identified via MS-based proteomics where acetylated peptides have been enriched using acetylated lysine specific antibodies. However, further work investigating endogenous stoichiometry (or site occupancy) of acetylated lysines has revealed that the vast majority are below 1%. Meaning, for most sites, less than 1% of the pool of a protein actually has an acetyl group on a particular lysine (see https://www.ncbi.nlm.nih.gov/pubmed/26358839 and https://www.ncbi.nlm.nih.gov/pubmed/24489116). This clearly calls into question the ability of acetylation to drastically alter the function of most of the proteins identified as ‘targets’.

A very interesting hypothesis is emerging, whereby many of the identified sites of acetylation are not mediated by the specific transfer of acetyl groups via acetyl-transferase enzymes in cells, but are direct acceptors of acetyl groups from reactive chemicals such as acetyl-CoA, or acetyl-phosphate (an earlier review can be found here https://www.ncbi.nlm.nih.gov/pubmed/24725594). This is termed, non-enzymatic, or chemical modification.

Intriguingly, this proximity-based direct modification process may not be restricted to non-enzymatic modification systems. In fact the majority of enzyme-catalysed cellular post-translational modifications involve highly reactive intermediates (such as thioester-bonded ubiquitin or ubiquitin-like modifiers to E1 or E2 enzymes), which can modify lysines in absence of the specificity-determining enzymes (E3 ligases). So it follows that ‘unintended’ modifications can occur for any biologically relevant post-translational modification simply by spatial proximity. This actually also fits with the acetylation site occupancy studies that showed (relatively) higher occupancy in proteins that are themselves involved in acetylation dynamics. Couple these theories with the exquisitely sensitive detection methods used in modern proteomics studies, and we have the potential to create huge lists of modification sites where the proportion with true biological relevance is unknown.

Where does this all fit in with this work describing post-translational modification of cellular proteins with CoA? Reviewing these data bearing the above in mind, it seems the simplest explanation is that non-enzymatic CoAlation occurs in cells when the redox potential has shifted to tip the balance in favour of reaction of CoA with cysteine thiols in proximal proteins. Removal of oxidising agents would allow the balance to revert to more reducing conditions, and so reversal of the CoAlation. The data presented in this paper support this idea as CoAlation is redox-dependent and ‘targets’ proteins that are known to interact with CoA in the cell.

In short, as with many of the published post-translational modification proteomes, much needs to be done to give biological credibility to sites of CoAlation. In particular occupancy calculations and protein-specific evidence that CoAlation regulates function in vivo, will go a long way to putting the notion of biological relevance beyond reasonable doubt. Until then we should consider the possibility that in many cases, post-translational modifications identified by modern methods have the potential to be the unintended consequence of interactions between reactive molecules and nearby proteins. It is worth noting that such a situation does not exclude biological relevance, but it makes finding any very challenging.

On 2017 May 16, Michael Tatham commented:

Is CoAlation biologically relevant or a non-functional by-product of the chemical reaction between CoA and cysteine thiols in proximal proteins under certain redox conditions?

Firstly, in my opinion the work described here is technically sound. The development of the specific antibody for CoA, and the mass spectrometric method to detect the modification on peptides are key tools in the analysis of any post-translational modification. However, there is a risk when using these super-sensitive methods, that one can detect vanishingly small amounts of modified peptides, which inevitably calls relevance into question. More specifically, modern mass-spectrometry based proteomics in combination with peptide-level enrichment of modified species has allowed us to identify modification sites in the order of tens of thousands for phosphorylation, ubiquitination, acetylation and SUMOylation (as of May 2017). For these fields, the onus of the researcher has very quickly shifted from identification of sites, to evidence for biological meaning. In short, the question is no longer “Which proteins?”, but “Why?”.

Taking acetylation as an example: Phosphositeplus (www.phosphosite.org) lists over 37000 acetylation sites, the majority identified via MS-based proteomics where acetylated peptides have been enriched using acetylated lysine specific antibodies. However, further work investigating endogenous stoichiometry (or site occupancy) of acetylated lysines has revealed that the vast majority are below 1%. Meaning, for most sites, less than 1% of the pool of a protein actually has an acetyl group on a particular lysine (see https://www.ncbi.nlm.nih.gov/pubmed/26358839 and https://www.ncbi.nlm.nih.gov/pubmed/24489116). This clearly calls into question the ability of acetylation to drastically alter the function of most of the proteins identified as ‘targets’.

A very interesting hypothesis is emerging, whereby many of the identified sites of acetylation are not mediated by the specific transfer of acetyl groups via acetyl-transferase enzymes in cells, but are direct acceptors of acetyl groups from reactive chemicals such as acetyl-CoA, or acetyl-phosphate (an earlier review can be found here https://www.ncbi.nlm.nih.gov/pubmed/24725594). This is termed, non-enzymatic, or chemical modification.

Intriguingly, this proximity-based direct modification process may not be restricted to non-enzymatic modification systems. In fact the majority of enzyme-catalysed cellular post-translational modifications involve highly reactive intermediates (such as thioester-bonded ubiquitin or ubiquitin-like modifiers to E1 or E2 enzymes), which can modify lysines in absence of the specificity-determining enzymes (E3 ligases). So it follows that ‘unintended’ modifications can occur for any biologically relevant post-translational modification simply by spatial proximity. This actually also fits with the acetylation site occupancy studies that showed (relatively) higher occupancy in proteins that are themselves involved in acetylation dynamics. Couple these theories with the exquisitely sensitive detection methods used in modern proteomics studies, and we have the potential to create huge lists of modification sites where the proportion with true biological relevance is unknown.

Where does this all fit in with this work describing post-translational modification of cellular proteins with CoA? Reviewing these data bearing the above in mind, it seems the simplest explanation is that non-enzymatic CoAlation occurs in cells when the redox potential has shifted to tip the balance in favour of reaction of CoA with cysteine thiols in proximal proteins. Removal of oxidising agents would allow the balance to revert to more reducing conditions, and so reversal of the CoAlation. The data presented in this paper support this idea as CoAlation is redox-dependent and ‘targets’ proteins that are known to interact with CoA in the cell.

In short, as with many of the published post-translational modification proteomes, much needs to be done to give biological credibility to sites of CoAlation. In particular occupancy calculations and protein-specific evidence that CoAlation regulates function in vivo, will go a long way to putting the notion of biological relevance beyond reasonable doubt. Until then we should consider the possibility that in many cases, post-translational modifications identified by modern methods have the potential to be the unintended consequence of interactions between reactive molecules and nearby proteins. It is worth noting that such a situation does not exclude biological relevance, but it makes finding any very challenging.