Research on Protein Acetylation Functions in Hyperthermophilic Archaea

Jingjie Academic Interpretation

Protein acetylation is one of the major post-translational modifications (PTMs), and lysine acetylation can occur in many eukaryotic and bacterial proteins. Nα-acetylation is a common type of post-translational modification in eukaryotes, involved in regulating protein interactions, localization, folding, and degradation. Nα-acetylation is catalyzed by N-terminal acetyltransferases (NAT) and lysine acetyltransferases (KAT), transferring the acetyl group from acetyl-CoA to the α-amino group of lysine to form Nα-acetylation. Currently, there are six types of NATs in humans, namely NatA -NatF. In addition, bacteria (such as Escherichia coli and Bacillus subtilis) and eukaryotic mitochondria also mediate protein acetylation through non-enzymatic pathways. Widespread Nα-acetylation has also been found in bacteria (for example, about 18% of proteins in Pseudomonas aeruginosa), with E. coli RimL, RimJ, and RimI being known bacterial NATs, but the roles of these bacterial NATs remain unclear.

The Huang Li research group at the Institute of Microbiology, Chinese Academy of Sciences, primarily engages in the study of extreme environment microorganisms, focusing on the genetic mechanisms and environmental adaptability of extreme thermophilic archaea. Recently, the international academic journal Mol Cell Proteomics published an online research paper titledFunctional insights into protein acetylation in the hyperthermophilic archaeon Sulfolobus islandicus, where researchers systematically studied the functions of protein acetylation in hyperthermophilic archaea using acetylation modification proteomics.

Compared to eukaryotes and bacteria, the study of protein acetylation in archaea is relatively limited. It is known that the protein acetyltransferase ARD1 in S. islandicus can catalyze Nα-acetylation, while the protein acetyltransferase Pat mediates Nε-acetylation modification of proteins. In this study, the authors performed Nε- and Nα-acetylation proteomics analysis on wild-type S. islandicus and mutants deficient in Pat or ARD1. The results showed that N-terminal acetylation of proteins accounted for about 44% of identified proteins, while internal lysine residue modifications accounted for 26% of the theoretical total of gene-encoded proteins. Sis Pat mediates a small portion of protein acetylation modifications, primarily involving acyl-CoA synthetase as a preferred substrate, possibly participating in metabolic regulation. Sis Ard1 exhibited broader substrate specificity and could mediate acetylation modifications of human N-terminal acetyltransferase NatA-NatF target proteins, potentially involved in biological processes including cell cycle control, DNA replication, and CRISPR-based immune responses. Jingjie Biology provided technical support for the quantitative analysis of acetylation modifications in this study.

Research Background

1. Loss of SisPat in S. islandicus (ΔSisPat) shows a growth rate similar to that of the parent strain.

The authors constructed a protein acetyltransferase Pat-deficient mutant strain (ΔSisPat) and compared the growth rates of ΔSisPat, the parent strain S. islandicus E233S, and the plasmid-mediated ΔSisPat overexpression strain (pSeSD-SisPat). It was found that ΔSisPat did not exhibit any growth defects (Figure 1A-B). The authors also conducted ITRAQ-based quantitative proteomics analysis on ΔSisPat and the parent strain. A total of 1,586 proteins were identified, with 1,543 being quantifiable. In cases where the fold change >1.5, only four proteins in ΔSisPat exhibited differential expression changes (Figure 1C).

Figure 1. Sispat gene deletion effects on S. islandicus growth and protein expression levels

2. Pat enzyme mediates a small portion of protein acetylation modifications in vivo

ΔSisPat and the parent strain underwent acetylation modification proteomics studies, revealing that lysine N-ε-acetylation sites are widely present in S. islandicus, with a total of 1,708 Nε-acetylation lysine sites identified on 684 proteins, of which 1,503 sites from 644 proteins were quantifiable. Compared to the parent strain, only 24 sites exhibited differential changes in the ΔSisPat strain (fold change >1.3), with nearly all sites showing downregulated acetylation modification levels. Six acyl-CoA synthetases in vivo are substrates for Sis Pat (Figure 2A), indicating that Sis Pat-mediated Nε-acetylation modifications of proteins primarily participate in maintaining cellular metabolic balance.Sis Pat-mediated acetylation modifications of acyl-CoA synthetase are conserved (Figure 2B-C).

Figure 2. Sis Pat-mediated acetylation modifications of acyl-CoA synthetase

3. SisArd1 is an important Nα-acetyltransferase with broad substrate specificity

The authors also constructed an Nα-acetyltransferase Ard1-deficient strain (ΔSisArd1), showing that Ard1 is essential for the growth of S. islandicus. Consistent with the growth phenotype of ΔSisArd1, the expression levels of proteins involved in cell division, cell cycle control, DNA replication, and purine synthesis were significantly reduced in the mutant. The acetylation modification proteomics results of ΔSisArd1 compared to the parent strain indicated that 158 Nα-acetylation modified proteins were identified in the parent strain (accounting for 44% of the total identified proteins). However, only 36 Nα-acetylation modified proteins were identified in the ΔSisArd1 strain (accounting for 11% of the total identified proteins) (Figure 3B-C).92% of Nα-acetylation modifications are mediated by SisArd1.SisArd1 exhibits broader substrate specificity. and can mediate acetylation modifications of human N-terminal acetyltransferase NatA-NatF target proteins.

Figure 3. Nα-acetylation modified peptides identified in the parent strain and ΔSisArd1 of S. islandicus

In summary, this study conducted proteomics and acetylation modification analysis on wild-type hyperthermophilic archaeon Sulfolobus islandicus, the protein acetyltransferase Pat-deficient mutant strain (ΔSisPat) and the Nα-acetyltransferase Ard1-deficient mutant strain (ΔSisArd1), discovering that archaea, like ordinary bacteria and eukaryotes, can undergo acetylation modifications of lysine Nε-amino and Nα-amino. This is the first report on the substrate specificity and potential roles of these two archaeal protein acetyltransferases.

References

Jingjing Cao, et al., 2019, Functional insights into protein acetylation in the hyperthermophilic archaeon Sulfolobus islandicus. Mol Cell Proteomics.