Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome

Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques

Forecasting deforestation and carbon emissions in tropical developing countries facing demographic expansion: A case study in Madagascar

Anthropogenic deforestation in tropical countries is responsible for a significant part of global carbon dioxide emissions in the atmosphere. To plan efficient climate change mitigation programs (such as REDD+, Reducing Emissions from Deforestation and forest Degradation), reliable forecasts of deforestation and carbon dioxide emissions are necessary. Although population density has been recognized as a key factor in tropical deforestation, current methods of prediction do not allow the population explosion that is occurring in many tropical developing countries to be taken into account. Here, we propose an innovative approach using novel computational and statistical tools, including R/GRASS scripts and the new phcfM R package, to model the intensity and location of deforestation including the effect of population density. We used the model to forecast anthropogenic deforestation and carbon dioxide emissions in five large study areas in the humid and spiny-dry forests of Madagascar. Using our approach, we were able to demonstrate that the current rapid population growth in Madagascar (+3.39% per year) will significantly increase the intensity of deforestation by 2030 (up to +1.17% per year in densely populated areas). We estimated the carbon dioxide emissions associated with the loss of aboveground biomass to be of 2.24 and 0.26 tons per hectare and per year in the humid and spiny-dry forest, respectively. Our models showed better predictive ability than previous deforestation models (the figure of merit ranged from 10 to 23). We recommend this approach to reduce the uncertainty associated with deforestation forecasts. We also underline the risk of an increase in the speed of deforestation in the short term in tropical developing countries undergoing rapid population expansion. (Résumé d'auteur

Augmented Cystine–Glutamate Exchange by Pituitary Adenylate Cyclase-activating Polypeptide Signaling via the VPAC1 Receptor

In the central nervous system, cystine import in exchange for glutamate through system xc- is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc- activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine–glutamate exchange via system xc-. In this study, 24-h treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc- function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc- inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc- activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine–glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc-. Furthermore, the potentiation of system xc- activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc- activity

Reversal of age-related learning deficiency by the vertebrate PACAP and IGF-1 in a novel invertebrate model of aging: the pond snail (Lymnaea Stagnalis)

With the increase of life span, nonpathological age-related memory decline is affecting an increasing number of people. However, there is evidence that age-associated memory impairment only suspends, rather than irreversibly extinguishes, the intrinsic capacity of the aging nervous system for plasticity (1). Here, using a molluscan model system, we show that the age-related decline in memory performance can be reversed by administration of the pituitary adenylate cyclase activating polypeptide (PACAP). Our earlier findings showed that a homolog of the vertebrate PACAP38 and its receptors exist in the pond snail (Lymnaea stagnalis) brain (2), and it is both necessary and instructive for memory formation after reward conditioning in young animals (3). Here we show that exogenous PACAP38 boosts memory formation in aged Lymnaea, where endogenous PACAP38 levels are low in the brain. Treatment with insulin-like growth factor-1, which in vertebrates was shown to transactivate PACAP type I (PAC1) receptors (4) also boosts memory formation in aged pond snails. Due to the evolutionarily conserved nature of these polypeptides and their established role in memory and synaptic plasticity, there is a very high probability that they could also act as “memory rejuvenating” agents in humans

Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan–protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan–protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan–protein–protein interactions, as well as a molecular-level understanding of those complexes

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

<div><p>P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the <i>Dmd^mdx</i> mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of <i>p2rx7</i> also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.</p></div

The epidemiology of invasive pneumococcal disease in the Canadian North from 1999 to 2010

Introduction . The International Circumpolar Surveillance network is a population-based surveillance system that collects data on invasive pneumococcal disease (IPD) in Northern Canada. A 7-valent pneumococcal conjugate vaccine was first introduced in some regions of Northern Canada in 2002, followed by 10-valent (2009) and 13-valent (PCV-13) vaccines (2010). A 23-valent polysaccharide (PPV-23) vaccine was first introduced in 1988 for special populations and adults aged 65 years and older. To describe the epidemiology in the context of pneumococcal vaccination programs, we analysed surveillance data from Northern Canada from 1999 to 2010. Methods . A standardized case report form capturing demographic and clinical information was completed for all IPD cases in Northern Canada meeting the national case definition. Isolates were sent to a reference laboratory for confirmation, serotyping and antimicrobial resistance testing. Both laboratory and epidemiological data were sent to the Public Health Agency of Canada for analysis. Population denominators were obtained from Statistics Canada. Results . From 1999 to 2010, 433 IPD cases were reported (average 36 cases per year). Incidence was greatest among infants aged &#60;2 years and among those aged 65 years and older, with an average annual incidence of 133 and 67 cases per 100,000 population, respectively. After a peak in incidence in 2008, rates among infants have declined. Incidence rates varied from 2 to 16 times greater, depending on the year, among Aboriginals compared to non-Aboriginals. Hospitalization was reported in 89% of all cases and the case fatality ratio was 6.0%. Clinical manifestations varied, with some patients reporting &#x003E;1 manifestation. Pneumonia was the most common (70%), followed by bacteremia/septicaemia (30%) and meningitis (8%). Approximately, 42% of cases aged &#60;2 years in 2009 and 2010 had serotypes covered by the PCV-13. In addition, the majority (89%) of serotypes isolated in cases aged 65 years and older were included in the PPV-23 vaccine. Conclusion . IPD continues to be a major cause of disease in Northern Canadian populations, with particularly high rates among infants and Aboriginals. Continued surveillance is needed to determine the impact of conjugate pneumococcal vaccine programs. Additional studies investigating factors that predispose infants and Aboriginal peoples would also be beneficial