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.