Arginine phosphorylation during spore germination in bacteria
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Bacillus Subtilis
SUBMITTER: 
Nicolas Nalpas
LAB HEAD: Boris Macek
PROVIDER: PXD011082 | Pride | 2022-03-02
REPOSITORIES: Pride
Similar Datasets
Project description:Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate of Mtb. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on several functionally diverse targets within the Msm proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species.
2022-09-30 | PXD032083 | Pride
Project description:Clostridioides difficile is the leading cause of intestinal nosocomial post-antibiotic infections in adults. During infection, the bacterium must rapidly respond and adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Recently, Hanks-type Serine/Threonine kinases (STKs) and phosphatases (STPs), have emerged as important players in bacterial cell signaling and pathogenicity. However, characterization of STKs targets in prokaryotes remained a difficult challenge. Here, we applied and adapted the TiO2 phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We have identified and quantified 2497 proteins representing 63,1% of the theoretical proteome. Among these proteins, 649 contained phosphorylated peptides with a localization probability >0,75. This pathogen encodes two STKs (PrkC and CD2148) and one associated phosphatase (STP), PrkC was shown to be crucial in the regulation of cell wall homeostasis, antibiotic resistance and cell division. Comparative phosphoproteomics of wild-type, prkC, CD2148, stp and double prkC CD2148 strains were performed in order to determinate STKs targets. 104 proteins were identified as specific substrates of PrkC, 46 of CD2148 and 94 of both kinases. Using a combination of in vivo and in vitro approaches, FtsK and Spo0A were validated as a direct target of PrkC. This study provides a detailed mapping of kinase-substrate relationships which may aid in the identification of new biomarkers and therapeutic targets.
2022-12-08 | PXD029827 | Pride
Project description:Background: Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined. Results: Here, we examined whether protein phospho-modifications occur during germination, the first step of exiting dormancy, thereby facilitating spore revival. Utilizing Bacillus subtilis as a model organism, we performed phosphoproteomic analysis to define the Ser/Thr/Tyr phosphoproteome of a reviving spore. The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism and spore-specific determinants. Quantitative comparison of dormant and germinating spore phosphoproteomes revealed phosphorylation dynamics, indicating that phospho-modifications could modulate protein activity during this cellular transition. Furthermore, by mutating select phosphorylation sites located within proteins representative of key biological processes, we established a functional connection between phosphorylation and the progression of spore revival. Conclusions: In this study we provide for the first time a phosphoproteomic view of a germinating bacterial spore. We show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.
2015-08-27 | PXD002559 | Pride
Project description:Proof-of-principle pull-down experiments using a YwlE trapping mutant phosphatase to selective capture arginine phosphorylated proteins. Phosphoarginine is an acid labile protein modification that was demonstrated to exist in bacteria and probably also in higher eukaryotes. Due to the high abundance of Ser, Thr, and Tyr modification, specific enrichment of this type of phosphorylation is required to detect this modification in complex samples and distinguish them from other protein phosphorylations. For this purpose, the phosphoarginine-specific phosphatase YwlE (G.stearothermophilus) was modified by introduction of mutations in catalytically-active amino acids C9 and D118, as well as the surface residue F39. The efficiency of these mutants to enrich arginine phosphorylated proteins from B. subtilis cell extracts was tested in comparison to a shotgun phosphoproteomics approach. Phosphatase trapping mutants were expressed in E.coli with a C-terminal histidine-tag. B.subtilis ywle cultures were grown in LB medium and heat shocked to induce arginine phosphorylation by the protein arginine kinase McsB. Cells were lysed under native conditions and incubated with the trapping mutant. Phosphorylated proteins were enriched by Ni2+/NTA chromatography to isolate the trapping mutant and bound substrates. Sample were prepared for bottom up proteomic analysis by reduction of disulfides, subsequent alkylation of free cysteines and tryptic digestion on the beads. Phosphopeptides were enriched from the resulting peptide mixtures using an optimized TiO2-based enrichment protocol and subsequently submitted to LC-MS/MS analysis.
2014-05-19 | PXD000560 | Pride
Project description:Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. We report that protein arginine phosphorylation plays a physiological significant role for the regulation of protein activity. We detected 121 arginine phospho-sites for 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidences that arginine phosphorylations are involved in the fine-tuned signal transduction of many critical cellular processes, such as protein degradation, motility, competence, stringent and stress response. Our results suggest that in B. subtilis the activity of a protein arginine phosphatase allows a fast regulation of protein activity by protein arginine kinases and that protein arginine phosphorylations play an important role as a reversible post-translational modification in bacteria.
2012-04-01 | GSE31249 | GEO
Project description:Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. We report that protein arginine phosphorylation plays a physiological significant role for the regulation of protein activity. We detected 121 arginine phospho-sites for 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidences that arginine phosphorylations are involved in the fine-tuned signal transduction of many critical cellular processes, such as protein degradation, motility, competence, stringent and stress response. Our results suggest that in B. subtilis the activity of a protein arginine phosphatase allows a fast regulation of protein activity by protein arginine kinases and that protein arginine phosphorylations play an important role as a reversible post-translational modification in bacteria. Cells were grown under vigorous agitation at 37 M-BM-0C in a defined medium (StM-CM-<lke et al., 1993, J Gen Microbiol 139, 2041-2045). Samples were taken at OD500 0.4 and 1h upon entry into stationary phase. Microarray hybridizations were performed with RNA from three biological replicates. The individual samples were labeled with Cy5; a reference pool containing equal amounts of RNA from all 10 samples was labeled with Cy3.
2012-03-31 | E-GEOD-31249 | biostudies-arrayexpress
Project description:Ever since the identification of the first protein-arginine kinase, McsB, in B. subtilis arginine phosphorylation gained more attention. However, the analysis of phosphorylations especially phosphormaidates comes along with several challenges. The sub-stoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis and the acid instability of phosphoramidates was long though to impede those enrichments. Further good spectral quality is required which can be reduced by the presence of neutral losses of phosphoric acid upon higher energy collision induced dissociation. Adaptation of common enrichment strategies to less acidic conditions and the use of electron-transfer dissociation allowed the identification of more than 200 pArg sites in B. subtilis and S. aureus so far. Here we show that pArg is stable enough for commonly used Fe3+-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for phosphoproteomics. By profiling a serine/ threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic phosphoarginine peptides we validated the presence and localization of arginine phosphorylation in S. aureus and lastly, we could show that Stp1 is influencing the arginine phosphoproteom without being a protein-arginine phosphatase.
2022-06-09 | PXD026981 | Pride
Project description:The formation of condensates in membraneless organelles is thought to be driven by protein phase separation. Arginine methylation and serine/threonine phosphorylation are important in the phase separation process, however these post-translational modifications are often present in intrinsically disordered regions that are difficult to analyse with standard proteomic techniques. Here we use a multi-protease and multi-MS/MS fragmentation approach, coupled with heavy methyl SILAC and phospho- or methyl-peptide enrichment, for the analysis of arginine methylation and serine/threonine phosphorylation, and to understand their co-occurrence in condensate-associated proteins. For Saccharomyces cerevisiae, we report a 50% increase in the known arginine methylproteome, involving 15 proteins that are almost all condensate-associated. Importantly, some of these proteins have arginine methylation on all predicted sites – providing evidence that this modification can be pervasive. We explored whether arginine methylated condensate-associated proteins are also phosphorylated, and found 12 such proteins to carry phosphoserine or phosphothreonine. In Npl3, Ded1 and Ssbp1, single peptides were found to carry both modifications, indicating a co-occurrence in close proximity and on the same protein molecule. We show that these co-modifications occur in regions of disorder and that arginine methylation is typically on basic regions of disorder. For phosphorylation, its association with charged regions of condensate-associated proteins was less consistent, although some regions with multisite phosphorylation sites were strongly acidic. We conclude that arginine-methylated proteins associated with condensates are typically co-modified with protein phosphorylation.
2021-09-17 | PXD022403 | Pride
Project description:Protein methyltransferases can recognise their substrates through linear sequence motifs and determination of these motifs is critical to understand methyltransferase mechanism, function and drug targeting. Here we describe MT-MAMS (methyltransferase motif analysis by mass spectrometry), a quantitative approach to characterise methyltransferase substrate recognition motifs and enzyme processivity. We validated MT-MAMS by application to lysine methyltransferase G9a, then determined the recognition motifs of Efm1 and major arginine methyltransferases Hmt1 and PRMT1.
2018-09-03 | PXD009515 | Pride
Project description:Obtaining a systems view of G protein-coupled receptor (GPCR) signaling in its native environment is the key in development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as model GPCR, we employed high-throughput phosphoproteomics to investigate downstream signaling induced by structurally diverse agonists in five mouse brain-regions. Through quantification of 50,000 different phosphosites, this approach yielded a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Pathway-selective agonists elicited differential dynamic phosphorylation of synaptic proteins, linking GPCR signaling to modulation of brain functions. We also discovered enrichment of mTOR pathway in agonists associated with aversion, a side effect. Consequently, mTOR inhibition during KOR activation abolished aversion, while preserving therapeutic analgesic and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.
2018-06-15 | PXD006637 | Pride