Alain J. Cozzone

Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis.

Publisher Summary This chapter discusses the chemical properties and separation of phosphoamino acids by thin-layer chromatography and electrophoresis. Phosphorylated amino acid residues in proteins are commonly classified into three main groups: (1) O -phosphates or O -phosphomonoesters, which are formed by phosphorylation of the hydroxyamino acids serine, threonine, and tyrosine; (2) N-phosphates or phosphoramidates, which are produced by phosphorylation of the basic amino acids arginine, histidine, and lysine; and (3) acylphosphates or phosphate anhydrides, which are generated by phosphorylation of the acidic amino acids aspartic acid and glutamic acid. Two types of intracellular phosphoproteins are known: enzymes that are intermediately phosphorylated, usually at their active sites, and proteins that are phosphorylated by protein kinases. Phosphorylated intermediates in enzymatic mechanisms are either phosphoramidates or acyl phosphates, but in some cases, such as phosphoglucomutase and alkaline phosphatase, the active site of the enzyme is modified at a serine residue.

A method to identily individual proteins in four different two-dimensional gel electrophoresis systems: Application to Escherichia coli ribosomal proteins

Abstract A new method to identify proteins in various two-dimensional polyacrylamide gel electrophoresis systems is described. Any previous purification of individual components is avoided by comparing the electrophoretic mobility of proteins in four different types of gels. Application of such a method to the particular case of Escherichia coli ribosomal proteins is presented.

Role of Protein Phosphorylation on Serine/Threonine and Tyrosine in the Virulence of Bacterial Pathogens

Bacterial pathogens have developed a diversity of strategies to interact with host cells, manipulate their behaviors, and thus to survive and propagate. During the process of pathogenesis, phosphorylation of proteins on hydroxyl amino acids (serine, threonine, tyrosine) occurs at different stages, including cell-cell interaction and adherence, translocation of bacterial effectors into host cells, and changes in host cellular structure and function induced by infection. The phosphorylation reactions are catalyzed in a reversible fashion by specific protein kinases and phosphatases that belong to either the invading bacterial cells or the infected eukaryotic host cells. Among the various virulence factors involved in bacterial pathogenesis, special attention has been paid recently to the cell wall components, exopolysaccharides. A major breakthrough has been made by showing the existence of a biological link between the activity of certain protein-tyrosine kinases/phosphatases and the production and/or transport of surface polysaccharides. In addition, genetic studies have revealed a key role played by some serine/threonine kinases in pathogenesis. Considering the structural organization and membrane topology of these different kinases, it can be envisaged that they operate as one-component systems in signal transduction pathways, in the form of single proteins containing input and output domains on the same polypeptide chain. From a general standpoint, the demonstration of a direct relationship between protein phosphorylation on serine/threonine/tyrosine and bacterial virulence represents a novel concept of great importance in deciphering the molecular and cellular mechanisms that underlie pathogenesis.

Autophosphorylation of the Escherichia coli Protein Kinase Wzc Regulates Tyrosine Phosphorylation of Ugd, a UDP-glucose Dehydrogenase

Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria. However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes. Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid. The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569. The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity. In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd. These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis. Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.

The Condensing Activities of the Mycobacterium tuberculosis Type II Fatty Acid Synthase Are Differentially Regulated by Phosphorylation

Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the β-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages.

Characterization of a Serine/Threonine Kinase Involved in Virulence of Staphylococcus aureus

Staphylococcus aureus is a common human cutaneous and nasal commensal and a major life-threatening pathogen. Adaptation to the different environments encountered inside and outside the host is a crucial requirement for survival and colonization. We identified and characterized a eukaryotic-like serine/threonine kinase with three predicted extracellular PASTA domains (SA1063, or Stk1) and its associated phosphatase (SA1062, or Stp1) in S. aureus. Biochemical analyses revealed that Stk1 displays autokinase activity on threonine and serine residues and is localized to the membrane. Stp1 is a cytoplasmic protein with manganese-dependent phosphatase activity toward phosphorylated Stk1. In-frame deletions of the stk1 and stp1 genes were constructed in S. aureus strain 8325-4. Phenotypic analyses of the mutants revealed reduced growth of the stk1 mutant in RPMI 1640 defined medium that was restored when adenine was added to the medium. Furthermore, the stk1 mutant displayed increased resistance to Triton X-100 and to fosfomycin, suggesting modifications in cell wall metabolism. The stk1 mutant was tested for virulence in a mouse pyelonephritis model and found to be strongly reduced for survival in the kidneys (approximately 2-log-unit decrease) compared to the parental strain. Renal histopathological analyses showed severe inflammatory lesions in mice infected with the parental S. aureus SH1000 strain, whereas the Deltastk1 mutant led to only minimal renal lesions. These results confirm the important role of Stk1 for full expression of S. aureus pathogenesis and suggest that phosphorylation levels controlled by stk1 are essential in controlling bacterial survival within the host.

Protein phosphorylation on tyrosine in bacteria

Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts. The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight. Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type. In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome. In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides. Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors. Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity.

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

ABSTRACT It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (Mw and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels.

Characterization of a bacterial gene encoding an autophosphorylating protein tyrosine kinase

Acinetobacter johnsonii harbors a protein tyrosine kinase activity that is able to catalyze autophosphorylation, like a number of eukaryotic tyrosine kinases. A biochemical and genetic analysis of this enzyme was performed. Maximum phosphorylation in vitro was obtained by incubating the kinase for 2 min at pH 7.0 in the presence of 5 mM magnesium chloride. In contrast to eukaryotic enzymes, no inhibitory effect of genistein and no phosphorylation of synthetic substrates such as poly (Glu80 Tyr20) or angiotensin II were observed. The analysis of the bacterial kinase by two-dimensional gel electrophoresis revealed the presence of at least five isoforms, all phosphorylated exclusively at tyrosine, which supports the concept that autophosphorylation occurs at multiple sites within the protein. The cloning and nucleotide sequencing of the gene encoding this kinase were achieved, which represents the first molecular characterization of a gene of this type in bacteria. An open reading frame of 2199 nucleotides encoding a protein of 82,373 Da was detected. The analysis of the deduced amino acid sequence suggested a possible involvement of the enzyme in cell recognition and bacterial pathogenicity. In addition, the cloning and sequencing of the region immediately upstream of the gene encoding the kinase revealed a novel open reading frame of 426 nucleotides encoding a phosphotyrosine protein phosphatase of 16,217 Da, which indicates that autophosphorylation on tyrosine is a physiologically reversible reaction.