Joanna Betts

Phosphorylation sites on Tau identified by nanoelectrospray mass spectrometry : Differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and p38, and glycogen synthase kinase-3β

Abstract: The stress‐activated kinases c‐Jun N‐terminal kinase (JNK) and p38 are members of the mitogen‐activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule‐associated protein tau, which becomes hyperphosphorylated in Alzheimers disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3β (GSK3β). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3β phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament‐tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3β are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimers disease.

The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo

Summary The function of the Mycobacterium tuberculosis eukaryotic‐like protein serine/threonine kinase PknG was investigated by gene knock‐out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C‐terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient‐depleted media. The PknG‐deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar‐containing molecules, GlcN‐Ins and mycothiol, which are derived from glutamate, were detected in the ΔpknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.

Differential expression of mycobacterial proteins following phagocytosis by macrophages

Mycobacterium tuberculosis resides within the macrophages of the host, but the molecular and cellular mechanisms of survival are poorly understood. Recent evidence suggests that the attenuated vaccine strain Mycobacterium bovis BCG is both a deletion and regulatory mutant, yet retains both its immunoprotective and intra-macrophage survival potential. In an attempt to define M. bovis BCG genes expressed during interaction with macrophages, the patterns of protein synthesis were examined by both one- and two-dimensional gel electrophoresis of BCG while inside the human leukaemic macrophage cell line THP-1. This study demonstrated that BCG expresses proteins while resident inside macrophages that are not expressed during in vitro growth in culture media or under conditions of heat shock. Western blotting analysis revealed that some of the differentially expressed proteins are specifically recognized by human M. tuberculosis-infected sera. Proteome analysis by two-dimensional electrophoresis and MS identified six abundant proteins that showed increased expression inside macrophages: 16 kDa alpha-crystallin (HspX), GroEL-1 and GroEL-2, a 31.7 kDa hypothetical protein (Rv2623), InhA and elongation factor Tu (Tuf). Identification of proteins by such a strategy will help elucidate the molecular basis of the attenuation and the vaccine potential of BCG, and may provide antigens that distinguish infection with M. tuberculosis from vaccination with BCG.

Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence

A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.

Signature Gene Expression Profiles Discriminate between Isoniazid-, Thiolactomycin-, and Triclosan-Treated Mycobacterium tuberculosis

ABSTRACT Genomic technologies have the potential to greatly increase the efficiency of the drug development process. As part of our tuberculosis drug discovery program, we used DNA microarray technology to profile drug-induced effects in Mycobacterium tuberculosis. Expression profiles of M. tuberculosis treated with compounds that inhibit key metabolic pathways are required as references for the assessment of novel antimycobacterial agents. We have studied the response of M. tuberculosis to treatment with the mycolic acid biosynthesis inhibitors isoniazid, thiolactomycin, and triclosan. Thiolactomycin targets the β-ketoacyl-acyl carrier protein (ACP) synthases KasA and KasB, while triclosan inhibits the enoyl-ACP reductase InhA. However, controversy surrounds the precise mode of action of isoniazid, with both InhA and KasA having been proposed as the primary target. We have shown that although the global response profiles of isoniazid and thiolactomycin are more closely related to each other than to that of triclosan, there are differences that distinguish the mode of action of these two drugs. In addition, we have identified two groups of genes, possibly forming efflux and detoxification systems, through which M. tuberculosis may limit the effects of triclosan. We have developed a mathematical model, based on the expression of 21 genes, which is able to perfectly discriminate between isoniazid-, thiolactomycin-, or triclosan-treated M. tuberculosis. This model is likely to prove invaluable as a tool to improve the efficiency of our drug development programs by providing a means to rapidly confirm the mode of action of thiolactomycin analogues or novel InhA inhibitors as well as helping to translate enzyme activity into whole-cell activity.

Use of gene expression profiling to identify a novel glucocorticoid sensitivity determining gene, BMPRII

Wide variation in glucocorticoid (Gc) sensitivity exists between individuals which may influence susceptibility to, and treatment response of, inflammatory diseases. To determine a genetic fingerprint of Gc sensitivity 100 healthy human volunteers were polarized into the 10% most Gc‐sensitive and 10% most Gc‐resistant following a low dose dexamethasone (0.25mg) suppression test. Gene expression profiling of primary lymphocytes identified the 98 most significantly Gc regulated genes. These genes were used to build a subnetwork of Gc signaling, with 54 genes mapping as nodes, and 6 non‐Gc regulated genes inferred as signaling nodes. Twenty four of the 98 genes showed a difference in Gc response in vitro dependent on the Gc sensitivity of their donor individuals in vivo. A predictive model was built using both partial least squares discriminate analysis and support vector machines that predicted donor glucocorticoid sensitivity with 87% accuracy. Discriminating genes included bone morphogenetic protein receptor, type II (BMPRII). Transfection studies showed that BMPRII modulated Gc action. These studies reveal a broad base of gene expression that predicts Gc sensitivity and determine a Gc signaling network in human primary T lymphocytes. Furthermore, this combined gene profiling, and functional analysis approach has identified BMPRII as a modulator of Gc signaling.—Donn, R., Berry, A., Stevens, A., Farrow, S., Betts, J., Stevens, R., Clayton, C., Wang, J., Warnock, L., Worthington, J., Scott, L., Graham, S., Ray, D. Use of gene expression profiling to identify a novel glucocorticoid sensitivity determining gene, BMPRII. FASEB J. 21, 402–414 (2007)

Transcriptomics and proteomics: Tools for the identification of novel drug targets and vaccine candidates for tuberculosis

The availability of the complete genome sequence of Mycobacterium tuberculosis has revolutionised many areas of tuberculosis research and facilitated functional genomics studies. Most notably, transcriptomics and proteomics have become important tools in gaining increased understanding of the biology of M. tuberculosis and offer the promise of being able to deliver novel drug targets and vaccine candidates.

Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3β identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes

In vitro phosphorylation of recombinant wild‐type 2N4R tau and FTDP‐17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3β (GSK3β) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates. Comparison of these peptide maps with those generated from wild‐type 1N4R tau isoform from which the phosphopeptide constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild‐type, P301L or V337M tau by GSK3β but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long‐range conformational effects on the structure of tau.

Protein expression in Mycobacterium tuberculosis differs with growth stage and strain type.

Abstract Different phenotypes are displayed by Mycobacterium tuberculosis (M. tuberculosis) strains, fuelling speculation that certain strains are “hypervirulent” and able to evade host defenses better than others. Furthermore, differential antigen expression by M. tuberculosis strains may explain why certain patients are susceptible to a repeat episode of tuberculosis. The objective of this study was to compare protein expression by M. tuberculosis H37Rv and clinical isolates in order to determine whether differential protein expression contributes to the different phenotypes expressed by these strains. Expression of α-crystallin, the antigen 85 complex, PstS-1, L-alanine dehydrogenase and the 65 kDa antigen was analysed by Western blotting and enzyme-linked immunosorbent assays, using mouse monoclonal antibodies. We found no significant difference in the growth rate of the M. tuberculosis strains in vitro, and although M. tuberculosis protein expression showed phase variation during growth, expression seemed to be qualitatively, but not quantitatively, conserved in the strains investigated. These results have potentially important implications for vaccine development and serodiagnosis.