Remarks
In recent years, the field of research on acetylation in bacteria has increased considerably, demonstrating its relevance in various cellular processes. Protein acetylation is a highly dynamic modification that occurs on short time scales (minutes to hours), where the acetylation levels are dictated by two closely related mechanisms, the addition and removal of the acyl group. (Baeza et al., 2020). This property allows the cell to respond quickly to different environmental changes by modifying the functionality of the protein to adjust metabolic processes.
The development of protein labeling and enrichment methods and technological advances in mass spectrometry for identifying and quantifying acetylated sites and proteins, have allowed the study of lysine acetylation in various organisms under different conditions. Thus, it has been shown that acetyl-lysines must result from one of the recognized acetylation mechanisms, whether enzymatic or non-enzymatic. Furthermore, it has been identified that the target proteins of acetylation participate in various cellular and metabolic processes, highlighting the acetylation of conserved lysines in central metabolism proteins. However, for many proteins, the biological relevance of this modification remains to be determined.
Another critical aspect that has yet to investigated deeply is the determination of the acetylation stoichiometry. Quantification of fold changes only sometimes has important biological significance. For example, if acetylation has functional consequences, such as loss of function, the modification should have a high stoichiometry. In comparison, a low percentage of acetylation may suggest a modification related to protein folding or the formation of a stable interaction (Carabetta & Cristea, 2016). So far, the techniques available for determining the stoichiometry have revealed that most acetylated proteins occur at low stoichiometry (0-10%), and in approximately 4% of acetylated sites a >20% stoichiometry is observed (Baeza et al., 2014; Weinert et al., 2017). The aforementioned indicates that only a small fraction of the acetylome is of physiological importance. Also, essential to consider that proteomic analysis is based on experiments under a particular condition and in isolated organisms, so it would be convenient to consider performing a comparative analysis under different conditions (temperature, carbon sources, among others) and their behavior in co-culture or symbiosis for a correct interpretation of the data.
Several functional monitoring studies are still being carried out usingin vitro assays, and although they allow us to know the effect of acetylation on the protein, only sometimes reflect the real consequence of this post-translational regulation process. Therefore, it is necessary to generate hybrid approaches that help us to identify and characterize not only the modified proteins but also in which metabolic pathways are involved and how this impacts the metabolism of the cell. Such approaches can integrate the analysis of proteomic, transcriptomic, and metabolomics data sets.
Incorporating synthetic biology can expand our understanding of how lysine acetylation regulates protein function. In this regard, genetic code expansion (GCE) has become essential for studing biological processes such as post-translational modifications. Rather than adding acetyl groups after protein translation, this approach relies on heterologous pairs of aminoacyl-tRNA-synthetases (aaRS) and its cognate tRNA from Methanosarcinaceae species that enable the co-translational incorporation of Nε-acetyllysine (AcK) in response to stop codon (Schmidt & Summerer, 2014). This approach has been used for some groups to evaluate the role of lysine acetylation using recombinant expressed proteins, finding that the enzymatic activity of several proteins is positively or negatively regulated. Once again, we believe this type of study should find an application for studies in bacteriain vivo .
Non-enzymatic acetylation is an inevitable consequence of the cell metabolic states. At high concentrations of AcP this mechanism is favored. However, it should be considered that this molecule can also be used as an acyl group donor for acetyltransferases catalyzing protein acetylation. Therefore, it cannot be ruled out that these mechanisms coexist. The roles of global acetylation and other acyl donors (succinyl-CoA, propionyl-CoA, and malonyl-CoA) for enzymatic or non-enzymatic protein acylation remain to be elucidated. Finally, it is essential to understand how lysine acetylation interferes with and cross-links with other post-translational modifications.