A Trigger Enzyme in Mycoplasma pneumoniae: Impact of the Glycerophosphodiesterase GlpQ on Virulence and Gene Expression

Mycoplasma pneumoniae is a causative agent of atypical pneumonia. The formation of hydrogen peroxide, a product of glycerol metabolism, is essential for host cell cytotoxicity. Phosphatidylcholine is the major carbon source available on lung epithelia, and its utilization requires the cleavage of deacylated phospholipids to glycerol-3-phosphate and choline. M. pneumoniae possesses two potential glycerophosphodiesterases, MPN420 (GlpQ) and MPN566. In this work, the function of these proteins was analyzed by biochemical, genetic, and physiological studies. The results indicate that only GlpQ is an active glycerophosphodiesterase. MPN566 has no enzymatic activity as glycerophosphodiesterase and the inactivation of the gene did not result in any detectable phenotype. Inactivation of the glpQ gene resulted in reduced growth in medium with glucose as the carbon source, in loss of hydrogen peroxide production when phosphatidylcholine was present, and in a complete loss of cytotoxicity towards HeLa cells. All these phenotypes were reverted upon complementation of the mutant. Moreover, the glpQ mutant strain exhibited a reduced gliding velocity. A comparison of the proteomes of the wild type strain and the glpQ mutant revealed that this enzyme is also implicated in the control of gene expression. Several proteins were present in higher or lower amounts in the mutant. This apparent regulation by GlpQ is exerted at the level of transcription as determined by mRNA slot blot analyses. All genes subject to GlpQ-dependent control have a conserved potential cis-acting element upstream of the coding region. This element overlaps the promoter in the case of the genes that are repressed in a GlpQ-dependent manner and it is located upstream of the promoter for GlpQ-activated genes. We may suggest that GlpQ acts as a trigger enzyme that measures the availability of its product glycerol-3-phosphate and uses this information to differentially control gene expression

Conservation, Variability and the Modeling of Active Protein Kinases

The human proteome is rich with protein kinases, and this richness has made the kinase of crucial importance in initiating and maintaining cell behavior. Elucidating cell signaling networks and manipulating their components to understand and alter behavior require well designed inhibitors. These inhibitors are needed in culture to cause and study network perturbations, and the same compounds can be used as drugs to treat disease. Understanding the structural biology of protein kinases in detail, including their commonalities, differences and modes of substrate interaction, is necessary for designing high quality inhibitors that will be of true use for cell biology and disease therapy. To this end, we here report on a structural analysis of all available active-conformation protein kinases, discussing residue conservation, the novel features of such conservation, unique properties of atypical kinases and variability in the context of substrate binding. We also demonstrate how this information can be used for structure prediction. Our findings will be of use not only in understanding protein kinase function and evolution, but they highlight the flaws inherent in kinase drug design as commonly practiced and dictate an appropriate strategy for the sophisticated design of specific inhibitors for use in the laboratory and disease therapy

Outer Membrane Integrity-Dependent Fluorescence of the Japanese Eel UnaG Protein in Live Escherichia coli Cells

Reporter genes are important tools in many biological disciplines. The discovery of novel reporter genes is relatively rare. However, known reporter genes are constantly applied to novel applications. This study reports the performance of the bilirubin-dependent fluorescent protein UnaG from the Japanese eel Anguilla japonicas in live Escherichia coli cells in response to the disruption of outer membrane (OM) integrity at low bilirubin (BR) concentrations. Using the E. coli wild-type strain MC4100, its isogenic OM-deficient mutant strain NR698, and different OM-active compounds, we show that BR uptake and UnaG fluorescence depend on a leaky OM at concentrations of 10 µM BR and below, while fluorescence is mostly OM integrity-independent at concentrations above 50 µM BR. We suggest that these properties of the UnaG–BR couple might be applied as a biosensor as an alternative to the OM integrity assays currently in use

J. Virol.

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C- terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase I (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin- dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. Four of the phosphorylation sites belong to integrin beta 4, a protein that mediates cell-matrix or cell-cell adhesion. The signature was validated in cross-validation and label switch experiments and in six independently profiled breast cancer cell lines. The study supports that the phosphorylation of integrin beta 4, as well as eight further proteins comprising the signature, are candidate biomarkers for predicting response to dasatinib in solid tumors. Furthermore, our results show that identifying predictive phosphorylation signatures from global, quantitative phosphoproteomic data is possible and can open a new path to discovering molecular markers for response prediction. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.016410, 651-668, 2012