Histidine phosphorylation in biological systems

Hsu

Molecular & Cellular Proteomics

et al. 2009

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD 50 increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD 50 increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. Molecular & Cellular Proteomics

Section: Resultssupporting

confidence: 79%

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

Hsu

Molecular & Cellular Proteomics

et al. 2009

“…In eukaryotes, phosphorylation takes place at serine, threonine, or tyrosine residues. Phosphorylation also occurs on histidine residues, however, phosphohistidine is highly labile and therefore rarely identified in phosphoproteomic studies [5]. Genomic sequencing has revealed that 2-3% of all eukaryotic genes are likely to code for protein kinases [6] and more than 100 human protein phosphatases have been predicted by genome annotation [7], emphasizing the ubiquitous role of protein phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

Proc. Natl. Acad. Sci. U.S.A.

“…Such lability would impair the transfer of the protein-DNA adduct across the cell membranes to the receiving cell. However, in proteins, the stability of the P-N bond is influenced by the protein environment, in particular by the side chains surrounding the phosphohistidine at the active site (37,38). For instance, a hydrogen bond to the nonphosphorylated imidazole nitrogen would make the phosphoramidate bond significantly less labile.…”

Section: Phosphoramidate Bond Chemistry and Consequences For Pmv158mentioning

confidence: 99%

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

Pluta

Boer

Lorenzo-Díaz

et al. 2017

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.histidine relaxase | antibiotic resistance | horizontal gene transfer | X-ray structure | Staphylococcus aureus A cquisition of exogenous genetic material by bacteria is achieved via conjugative DNA transfer of mobile genetic elements, such as plasmids and especially integrative and conjugative elements and integrative and mobilizable elements (1). Such processes of horizontal gene transfer (HGT) are considered a strong driving force in bacterial evolution and in the ability of bacteria to colonize different niches (2). In addition to permitting the rapid evolution of the bacterial pangenome, HGT is involved in the acquisition of genetic traits that may confer selective advantages to the recipient bacteria, including antibiotic resistance (3). This is particularly important when resistance genes encoded by mobile elements are spread explosively among bacteria in hospitals, posing a serious threat to public health systems (www.cdc.gov/drugresistance/threat-report-2013; www. who.int/drugresistance/documents/surveillancereport/en/). Thus, the so-called mobilome (4) participates in the spread of antibiotic resistance, which is expected to cause 10 million casualties annually by 2050, and the consequent huge economic burden (amr-review.org/sites/default/files/Report-52.15.pdf). This has generated a unanimous call for new approaches to deal with infectious diseases caused by pathogenic bacteria (5).A main performer in HGT is the protein relaxase, a topoisomeraselike enzyme that cleaves supercoiled plasmid DNA in a strandand sequence-specific manner and ligates it after cell-to-cell transfer. Relaxases start DNA transfer by conjugation on recognition of their target DNA, the origin of transfer (oriT), on which they mediate generation of a hairpin-loop structure that leaves the dinucleotide to be cleaved (the nic site) in a singlestranded (ss) DNA configuration (Fig. 1B) (6, 7). On the oriT, the relaxase assembles with other proteins p...