Acetylome of acinetobacter baumannii SK17 reveals a highly-conserved modification of histone-like protein HU

Lysine acetylation is a prevalent post-translational modification in both eukaryotes and prokaryotes. Whereas this modification is known to play pivotal roles in eukaryotes, the function and extent of this modification in prokaryotic cells remain largely unexplored. Here we report the acetylome of a pair of antibiotic-sensitive and -resistant nosocomial pathogen Acinetobacter baumannii SK17-S and SK17-R. A total of 145 lysine acetylation sites on 125 proteins was identified, and there are 23 acetylated proteins found in both strains, including histone-like protein HU which was found to be acetylated at Lys13. HU is a dimeric DNA-binding protein critical for maintaining chromosomal architecture and other DNA-dependent functions. To analyze the effects of site-specific acetylation, homogenously Lys13-acetylated HU protein, HU(K13ac) was prepared by genetic code expansion. Whilst not exerting an obvious effect on the oligomeric state, Lys13 acetylation alters both the thermal stability and DNA binding kinetics of HU. Accordingly, this modification likely destabilizes the chromosome structure and regulates bacterial gene transcription. This work indicates that acetyllysine plays an important role in bacterial epigenetics

Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

Abstract Background Phosphorylation of amino acid residues on proteins is an important and common post-translational modification in both eukaryotes and prokaryotes. Most research work has been focused on phosphorylation of serine, threonine or tyrosine residues, whereas phosphorylation of other amino acids are significantly less clear due to the controversy on their stability under standard bioanalytical conditions. Results Here we applied a shotgun strategy to analyze the histidine and aspartate phosphorylations in different microbes. Our results collectively indicate that histidine and aspartate phosphorylations frequently occur also in proteins that are not part of the two-component systems. Noticeably, a number of the modified proteins are pathogenesis-related or essential for survival in host. These include the zinc ion periplasmic transporter ZnuA in Acinetobacter baumannii SK17, the multidrug and toxic compound extrusion (MATE) channel YeeO in Klebsiella pneumoniae NTUH-K2044, branched amino acid transporter AzlC in Vibrio vulnificus and the RNA-modifying pseudouridine synthase in Helicobacter pylori. Conclusions In summary, histidine and aspartate phosphorylation is likely to be ubiquitous and to take place in proteins of various functions. This work also sheds light into how these functionally important proteins and potential drug targets might be regulated at a post-translational level

Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation

Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change

Additional file 2: Table S1. of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

Phospho probabilities, process method, leading protein identifier, UniProt No. (if available), protein description, and protein functional class of the identified phosphopeptides (PDF 539 kb)

Additional file 1: of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

MS/MS Spectra. Spectra of the identified phosphopeptides (PDF 4714 kb)

Additional file 3: Figure S1. of Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance

The relative abundances of amino acids between −10 and +10 positions of the phosphorylated or non-phosphorylated His/Asp (PDF 377 kb)

Phosphoproteomics and Bioinformatics Analyses Reveal Key Roles of GSK-3 and AKAP4 in Mouse Sperm Capacitation

Abstract: Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3↵ expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3↵ during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed