Pyrroloquinoline quinone biogenesis: characterization of PqqC and its H84N and H84A active site variants

Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial vitamin that serves as a cofactor in numerous alcohol dehydrogenases. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF, and proceeds by an unknown pathway. The protein encoded by pqqC catalyzes the final step of PQQ formation, which involves a ring closure and an overall eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) in the absence of a redox-active metal or cofactor. A recent crystal structure has implicated numerous PQQ-PqqC interactions [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918]. To investigate the mechanism of the PqqC reaction, the active site residue His84 has been mutated to H84A and H84N, and the kinetic and spectroscopic properties have been compared to each other and the wild-type enzyme using aerobic and anaerobic conditions. Both mutants form PQQ under aerobic conditions with rate constants of 0.09 min-1 and 0.056 min-1 relative to 0.34 min-1 for the wild-type enzyme. In addition to the initial E-AHQQ complex (532-536 nm) and the product E-PQQ complex (346-366 nm), a number of spectral intermediates are observed between 316 and 344 nm. The anaerobic reaction is particularly informative, showing that while mixing of H84N with AHQQ leads to a 344 nm intermediate, this is unable to proceed to a final 318 nm species; by contrast H84A forms the 344 nm species as a precursor to the 318 nm species. In the context of the proposed chemical mechanism for PqqC [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918], we assign the 344 nm intermediate to a quinoid species and the 318 nm intermediate to an initial quinol species. The proposed role of H84 is as a proton donor to the oxyanion of the quinoid species such that subsequent C-H bond cleavage can occur to form a monoanionic quinol. In the absence of a proton donor (as occurs in H84N), the normal reaction path is precluded as this would require formation of an unstable, dianionic species. Unlike H84N, H84A appears to be small enough to allow entry of active site water, which is postulated to adopt the role of active site proton donor.     

PqqD Is a Novel Peptide Chaperone That Forms a Ternary Complex with the Radical S-Adenosylmethionine Protein PqqE in the Pyrroloquinoline Quinone Biosynthetic Pathway

Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and K_D were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the K_D, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (K_D ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the K_D for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein.     

tigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities

Background

To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies.

Results

Here, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect.

Conclusion

With the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-327) contains supplementary material, which is available to authorized users.     

Effect of CpG oligonucleotides on vaccine-induced B cell memory

Adding synthetic oligodeoxynucleotides containing unmethylated CpG motifs to Anthrax vaccine adsorbed (AVA, the licensed human vaccine) increases the speed and magnitude of the resultant Ab response. Ab titers persist in the protective range for >1 year, significantly longer than in animals vaccinated with AVA alone. Unexpectedly, a majority of mice immunized with CpG-adjuvanted AVA maintained resistance to anthrax infection even after their Ab titers had declined into the subprotective range. The survival of these animals was mediated by the de novo production of protective Abs by high affinity memory B cells re-stimulated immediately after challenge. Thus, a previously unrecognized benefit of CpG oligodeoxynucleotides adjuvants is their ability to expand the long-lived memory B cell population. Current findings demonstrate that CpG-adjuvanted AVA mediates protection both by stimulating a strong/persistent serum Ab response and by generating a high-affinity long-lived pool of memory B cells.     

Radiographic joint damage in rheumatoid arthritis is associated with differences in cartilage turnover and can be predicted by serum biomarkers: an evaluation from 1 to 4 years after diagnosis

Introduction  

The objective of this study was to determine whether serum biomarkers for degradation and synthesis of the extracellular matrix of cartilage are associated with, and can predict, radiographic damage in patients with rheumatoid arthritis (RA).     

An atlas of human kinase regulation

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision‐making has been limited by the small number of simultaneously monitored phospho‐regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co‐regulation along the conditions predicts kinase–complex and kinase–substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation‐based signaling and the necessary context to better understand kinase‐driven decision‐making.     

Suppressive oligodeoxynucleotides inhibit Th1 differentiation by blocking IFN-gamma- and IL-12-mediated signaling

Repetitive TTAGGG motifs present at high frequency in mammalian telomeres can suppress Th1-mediated immune responses. Synthetic oligonucleotides (ODN) containing TTAGGG motifs mimic this activity and have proven effective in the prevention/treatment of certain Th1-dependent autoimmune diseases. This work explores the mechanism by which suppressive ODN block the induction of Th1 immunity. Findings indicate that these ODN inhibit IFN-gamma-induced STAT1 phosphorylation and IL-12-induced STAT3 and STAT4 phosphorylation. As a result, T-bet expression is reduced as is the maturation of naive CD4+ cells into Th1 effectors. These changes indirectly support the generation of Th2-dominated immune responses. Suppressive ODN may thus represent a novel approach to influence the Th1:Th2 balance in vivo.     

Characteristics of B cell proliferation and activation in murine AIDS

A syndrome characterized by lymphadenopathy, hypergammaglobulinemia, and immunodeficiency develops in C57BL/6 mice inoculated with LP-BM5 murine leukemia viruses. By studying the number and antigenic specificity of B cells activated in the course of this disease, we found that a series of reproducible changes in the humoral immune system were induced by retroviral infection. The rate of B cell proliferation and the proportion of B cells activated to secrete Ig increased by nearly 10-fold at 4 wk post inoculation. B cells producing antibodies reactive with a panel of three conventional Ag and five autoantigens were stimulated simultaneously and proportionally to secrete, demonstrating that such activation was polyclonal in nature. At 12 wk post infection, the number of Ig-secreting B cells continued to rise and significant hypergammaglobulinemia developed. At 16 wk post infection, immunostimulation gave way to immunosuppression, as evidenced by a slight decline in the number of Ig-secreting lymphocytes and a sharp reduction in the concentration of serum antibody. At this time, the B cell repertoires of infected mice diverged markedly from those of uninfected animals. These changes are comparable to those found in some patients infected with HIV, and provide a useful model to study the association between retroviral infection and regulatory abnormalities of the humoral immune system.