Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation,modelling and conformational changes

Recombinant FlagHis₆ tagged Human P2X1 receptors expressed in HEK293 cells were purified, digested with trypsin and analysed by mass spectroscopy (96% coverage following de‐glycosylation and reduction). The receptor was basally phosphorylated at residues S387, S388 and T389 in the carboxyl terminus, a triple alanine mutant of these residues had a modest ~ 25% increase in current amplitude and recovery from desensitization. Chemical modification showed that intracellular lysine residues close to the transmembrane domains and the membrane stabilization motif are accessible to the aqueous environment. The membrane‐impermeant cross‐linking reagent 3,3′‐Dithiobis (sulfosuccinimidylpropionate) (DTSSP) reduced agonist binding and P2X1 receptor currents by > 90%, and modified lysine residues were identified by mass spectroscopy. Mutation to remove reactive lysine residues around the ATP‐binding pocket had no effect on inhibtion of agonist evoked currents following DTSSP. However, agonist evoked currents were ~ 10‐fold higher than for wild type following DTSSP treatment for mutants K199R, K221R and K199R‐K221R. These mutations remove reactive residues distant from the agonist binding pocket that are close enough to cross‐link adjacent subunits. These results suggest that conformational change in the P2X1 receptor is required for co‐ordination of ATP action.     

Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses

Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure-function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H(+)-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses.     

Developmental and wound-, cold-, desiccation-, ultraviolet-B-stress-induced modulations in the expression of the petunia zinc finger transcription factor gene ZPT2-2

The ZPT2-2 gene belongs to the EPF gene family in petunia (Petunia hybrida), which encodes proteins with TFIIIA-type zinc-finger DNA-binding motifs. To elucidate a possible function for ZPT2-2, we analyzed its pattern of expression in relation to different developmental and physiological stress signals. The activity of the ZPT2-2 promoter was analyzed using a firefly luciferase (LUC) reporter gene, allowing for continuous measurements of transgene activity in planta. We show that ZPT2-2::LUC is active in all plant tissues, but is strongly modulated in cotyledons upon germination, in leaves in response to desiccation, cold treatment, wounding, or ultraviolet-B light, and in petal tissue in response to pollination of the stigma. Analysis of mRNA levels indicated that the modulations in ZPT2-2::LUC expression reflect modulations in endogenous ZPT2-2 gene expression. The change in ZPT2-2::LUC activity by cold treatment, wounding, desiccation, and ultraviolet-B light suggest that the phytohormones ethylene and jasmonic acid are involved in regulating the expression of ZPT2-2. Although up-regulation of expression of ZPT2-2 can be blocked by inhibitors of ethylene perception, expression in plants is not induced by exogenously applied ethylene. The application of jasmonic acid does result in an up-regulation of gene activity and, thus, ZPT2-2 may play a role in the realization of the jasmonic acid hormonal responses in petunia.     

Mitochondrial mismatch analysis is insensitive to the mutational process

Mismatch distributions are histograms showing the pattern of nucleotide (or restriction) site differences between pairs of individuals in a sample. They can be used to test hypotheses about the history of population size and subdivision (if selective neutrality is assumed) or about selection (if a constant population size is assumed). Previous work has assumed that mutations never strike the same site twice, an assumption that is called the model of infinite sites. Fortunately, the results are surprisingly robust even when this assumption is violated. We show here that (1) confidence regions inferred using the infinite- sites model differ little from those inferred using a model of finite sites with uniform site-specific mutation rates, and (2) even when site- specific mutation rates follow a gamma distribution, confidence regions are little changed until the gamma shape parameter falls well below its plausible range, to roughly 0.01. In addition, we evaluate and reject the proposition that mismatch waves are produced by pooling data from several subdivisions of a structured population.      

Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess ≤0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Effects of exercise and poor indoor air quality on learning,memory and blood IGF-1 in adolescent mice

It is known that regular aerobic exercise enhances cognitive functions and increases blood insulin-like growth factor 1 (IGF-1) levels. People living in urban areas spend most of their time indoors and indoor air quality can affect health. We investigated the effects of aerobic exercise in poor and good air quality environments on hippocampus and prefrontal cortex (PFC) neurons, anxiety, and spatial learning and memory in adolescent mice. Poor air quality impaired spatial learning and memory; exercise did not affect learning or memory impairment. Exercise in a good air quality environment improved spatial learning and memory. Poor air quality increased apoptosis in the hippocampus and PFC. Both exercised and sedentary groups living in a poor air quality environment had lower serum IGF-1 levels than those living in a good air quality environment. Living in a poor air quality environment has negative effects on the hippocampus, PFC and blood IGF-1 levels in adolescent mice, but exercise did not alter the negative effects of poor air quality.     

Anxiety- and depression-like behavior are correlated with leptin and leptin receptor expression in prefrontal cortex of streptozotocin-induced diabetic rats

Anxiety and depression are common in diabetics. Diabetes also may cause reduced leptin levels in the blood. We investigated the relation between diabetes induced anxiety- and depression-like behavior, and leptin and leptin receptor expression levels in diabetic rats. The anxiety- and depression-like behaviors of rats were assessed 4 weeks after intraperitoneal injection of streptozotocin. Diabetic rats exhibited greater anxiety-like behavior; they spent more time in closed branches of the elevated plus maze test and less time in the center cells of the open field arena. Increased depression-like behavior was observed in diabetic rats using the Porsolt swim test. Prefrontal cortex (PFC), blood leptin levels and PFC neuron numbers were decreased, and leptin receptor expression and apoptosis were increased in diabetic rats. Blood corticosterone levels also were increased in diabetic rats. These results indicate that reduction of leptin up-regulates leptin receptor expression and may affect PFC neurons, which eventually triggers anxiety- and depression-like behaviors in diabetic rats.     

The Role of zinc in the disulphide stress-regulated anti-sigma factor RsrA from Streptomyces coelicolor

The regulation of disulphide stress in actinomycetes such as Streptomyces coelicolor is known to involve the zinc-containing anti-sigma factor RsrA that binds and inactivates the redox-regulated sigma factor sigmaR. However, it is not known how RsrA senses disulphide stress nor what role the metal ion plays. Using in vitro assays, we show that while zinc is not required for sigmaR binding it is required for functional anti-sigma factor activity, and that it plays a critical role in modulating the reactivity of RsrA cysteine thiol groups towards oxidation. Apo-RsrA is easily oxidised and, while the Zn-bound form is relatively resistant, the metal ion is readily expelled when the protein is treated with strong oxidants such as diamide. We also show, using a combination of proteolysis and mass spectrometry, that the first critical disulphide to form in RsrA involves Cys11 and one of either Cys41 or Cys44, all previously implicated in metal binding. Circular dichroism spectroscopy was used to follow structural changes during oxidation of RsrA, which indicated that concomitant with formation of this critical disulphide bond is a major restructuring of the protein where its alpha-helical content increases. Our data demonstrate that RsrA can only bind sigmaR in the reduced state and that this state is stabilised by zinc. Redox stress induces disulphide bond formation amongst zinc-ligating residues, expelling the metal ion and stabilising a structure incapable of binding the sigma factor.