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

Urotensin receptor in GtoPdb v.2023.1

The urotensin-II (U-II) receptor (UT, nomenclature as agreed by the NC-IUPHAR Subcommittee on the Urotensin receptor [26, 36, 94]) is activated by the endogenous dodecapeptide urotensin-II, originally isolated from the urophysis, the endocrine organ of the caudal neurosecretory system of teleost fish [7, 93]. Several structural forms of U-II exist in fish and amphibians [94]. The goby orthologue was used to identify U-II as the cognate ligand for the predicted receptor encoded by the rat gene gpr14 [2, 20, 63, 69, 72]. Human urotensin-II, an 11-amino-acid peptide [20], retains the cyclohexapeptide sequence of goby U-II that is thought to be important in ligand binding [61, 53, 10]. This sequence is also conserved in the deduced amino-acid sequence of rat urotensin-II (14 amino-acids) and mouse urotensin-II (14 amino-acids), although the N-terminal is more divergent from the human sequence [19]. A second endogenous ligand for the UT has been discovered in rat [86]. This is the urotensin II-related peptide, an octapeptide that is derived from a different gene, but shares the C-terminal sequence (CFWKYCV) common to U-II from other species. Identical sequences to rat urotensin II-related peptide are predicted for the mature mouse and human peptides [32]. UT exhibits relatively high sequence identity with somatostatin, opioid and galanin receptors [94]. The urotensinergic system displays an unprecedented repertoire of four or five ancient UT in some vertebrate lineages and five U-II family peptides in teleost fish [91]

Urotensin receptor in GtoPdb v.2021.3

The urotensin-II (U-II) receptor (UT, nomenclature as agreed by the NC-IUPHAR Subcommittee on the Urotensin receptor [26, 36, 93]) is activated by the endogenous dodecapeptide urotensin-II, originally isolated from the urophysis, the endocrine organ of the caudal neurosecretory system of teleost fish [7, 92]. Several structural forms of U-II exist in fish and amphibians [93]. The goby orthologue was used to identify U-II as the cognate ligand for the predicted receptor encoded by the rat gene gpr14 [2, 20, 63, 69, 72]. Human urotensin-II, an 11-amino-acid peptide [20], retains the cyclohexapeptide sequence of goby U-II that is thought to be important in ligand binding [61, 53, 10]. This sequence is also conserved in the deduced amino-acid sequence of rat urotensin-II (14 amino-acids) and mouse urotensin-II (14 amino-acids), although the N-terminal is more divergent from the human sequence [19]. A second endogenous ligand for the UT has been discovered in rat [86]. This is the urotensin II-related peptide, an octapeptide that is derived from a different gene, but shares the C-terminal sequence (CFWKYCV) common to U-II from other species. Identical sequences to rat urotensin II-related peptide are predicted for the mature mouse and human peptides [32]. UT exhibits relatively high sequence identity with somatostatin, opioid and galanin receptors [93]

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

Urotensin receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The urotensin-II (U-II) receptor (UT, nomenclature as agreed by the NC-IUPHAR Subcommittee on the Urotensin receptor [26, 36, 89]) is activated by the endogenous dodecapeptide urotensin-II, originally isolated from the urophysis, the endocrine organ of the caudal neurosecretory system of teleost fish [7, 88]. Several structural forms of U-II exist in fish and amphibians. The goby orthologue was used to identify U-II as the cognate ligand for the predicted receptor encoded by the rat gene gpr14 [20, 62, 68, 70]. Human urotensin-II, an 11-amino-acid peptide [20], retains the cyclohexapeptide sequence of goby U-II that is thought to be important in ligand binding [53, 11]. This sequence is also conserved in the deduced amino-acid sequence of rat urotensin-II (14 amino-acids) and mouse urotensin-II (14 amino-acids), although the N-terminal is more divergent from the human sequence [19]. A second endogenous ligand for the UT has been discovered in rat [83]. This is the urotensin II-related peptide, an octapeptide that is derived from a different gene, but shares the C-terminal sequence (CFWKYCV) common to U-II from other species. Identical sequences to rat urotensin II-related peptide are predicted for the mature mouse and human peptides [32]. UT exhibits relatively high sequence identity with somatostatin, opioid and galanin receptors [89]

VIP and PACAP receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Vasoactive Intestinal Peptide Receptors [64, 65]) are activated by the endogenous peptides VIP, PACAP-38, PACAP-27, peptide histidine isoleucineamide (PHI), peptide histidine methionineamide (PHM) and peptide histidine valine (PHV). VPAC1 and VPAC2 receptors display comparable affinity for the PACAP peptides, PACAP-27 and PACAP-38, and VIP, whereas PACAP-27 and PACAP-38 are >100 fold more potent than VIP as agonists of most isoforms of the PAC1 receptor. However, one splice variant of the human PAC1 receptor has been reported to respond to PACAP-38, PACAP-27 and VIP with comparable affinity [29]. PG 99-465 [115] has been used as a selective VPAC2 receptor antagonist in a number of physiological studies, but has been reported to have significant activity at VPAC1 and PAC1 receptors [35]. The selective PAC1 receptor agonist maxadilan, was extracted from the salivary glands of sand flies (Lutzomyia longipalpis) and has no sequence homology to VIP or the PACAP peptides [116]. Two deletion variants of maxadilan, M65 [180] and Max.d.4 [117] have been reported to be PAC1 receptor antagonists, but these peptides have not been extensively characterised

VIP and PACAP receptors in GtoPdb v.2023.1

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Vasoactive Intestinal Peptide Receptors [65, 66]) are activated by the endogenous peptides VIP, PACAP-38, PACAP-27, peptide histidine isoleucineamide (PHI), peptide histidine methionineamide (PHM) and peptide histidine valine (PHV). VPAC1 and VPAC2 receptors display comparable affinity for the PACAP peptides, PACAP-27 and PACAP-38, and VIP, whereas PACAP-27 and PACAP-38 are &gt;100 fold more potent than VIP as agonists of most isoforms of the PAC1 receptor. However, one splice variant of the human PAC1 receptor has been reported to respond to PACAP-38, PACAP-27 and VIP with comparable affinity [30]. PG 99-465 [117] has been used as a selective VPAC2 receptor antagonist in a number of physiological studies, but has been reported to have significant activity at VPAC1 and PAC1 receptors [36]. The selective PAC1 receptor agonist maxadilan, was extracted from the salivary glands of sand flies (Lutzomyia longipalpis) and has no sequence homology to VIP or the PACAP peptides [118]. Two deletion variants of maxadilan, M65 [183] and Max.d.4 [119] have been reported to be PAC1 receptor antagonists, but these peptides have not been extensively characterised

When kinases meet mathematics: the systems biology of MAPK signalling

The mitogen activated protein kinase/extracellular signal regulated kinase pathway regulates fundamental cellular function such as cell proliferation, survival, differentiation and motility, raising the question how these diverse functions are specified and coordinated. They are encoded through the activation kinetics of the pathway, a multitude of feedback loops, scaffold proteins, subcellular compartmentalisation, and crosstalk with other pathways. These regulatory motifs alone or in combination can generate a multitude of complex behaviour. Systems biology tries to decode this complexity through mathematical modelling and prediction in order to gain a deeper insight into the inner works of signalling networks

Human and environmental controls over aboveground carbon storage in Madagascar

Background: Accurate, high-resolution mapping of aboveground carbon density (ACD, Mg C ha-1) could provide insight into human and environmental controls over ecosystem state and functioning, and could support conservation and climate policy development. However, mapping ACD has proven challenging, particularly in spatially complex regions harboring a mosaic of land use activities, or in remote montane areas that are difficult to access and poorly understood ecologically. Using a combination of field measurements, airborne Light Detection and Ranging (LiDAR) and satellite data, we present the first large-scale, high-resolution estimates of aboveground carbon stocks in Madagascar. Results: We found that elevation and the fraction of photosynthetic vegetation (PV) cover, analyzed throughout forests of widely varying structure and condition, account for 27-67 % of the spatial variation in ACD. This finding facilitated spatial extrapolation of LiDAR-based carbon estimates to a total of 2,372,680 ha using satellite data. Remote, humid sub-montane forests harbored the highest carbon densities, while ACD was suppressed in dry spiny forests and in montane humid ecosystems, as well as in most lowland areas with heightened human activity. Independent of human activity, aboveground carbon stocks were subject to strong physiographic controls expressed through variation in tropical forest canopy structure measured using airborne LiDAR. Conclusions: High-resolution mapping of carbon stocks is possible in remote regions, with or without human activity, and thus carbon monitoring can be brought to highly endangered Malagasy forests as a climate-change mitigation and biological conservation strategy

Mise en évidence de protéines révélant une réponse au stress adaptative divergente entre les espèces Dreissena polymorpha et Dreissena rostriformis bugensis

International audienceZebra mussels Dreissena polymorpha, are bivalve molluscs used in ecotoxicology, as a sentinel species with, among other things, a strong bioaccumulation capacity. The quagga mussel, Dreissena rostriformis bugensis, has more recently colonized Western Europe. Also invasive, it competes with zebra mussels for habitats and, in some areas, has completely replaced it. Its use as a sentinel species is envisaged but requires understanding the mechanisms involved during the adaptive stress response and comparing them to those of the zebra mussel, which are better characterized. With this in mind, an exposure of the two species to a classical contaminant was performed to compare their responses. The individuals were exposed to a concentration of 100 μg / L of cadmium for 7 days, and then the gill proteins were separated by two-dimensional electrophoresis and the variable abundance proteoforms were identified by mass spectrometry. The functional analysis reveals promising elements for the study of the adaptive response in both species. A difference of expression of actors of the energy metabolism pathways points a physiological difference, with a reallocation of energy as well as the appearance of truncated proteins.Les moules zébrées, Dreissena polymorpha sont des mollusques bivalves utilisés en écotoxicologie comme espèces sentinelles du fait, entre autre, de leur capacité de bioaccumulation. La moule quagga, Dreissena rostriformis bugensis, a colonisé plus récemment l'Europe occidentale. Egalement invasive, elle est en compétition avec la moule zébrée pour les habitats et, dans certaines zones, l'a complètement remplacée. Son utilisation en tant qu'espèce sentinelle est envisagée mais nécessite de comprendre les mécanismes mis en jeu au cours de la réponse adaptative au stress et de les comparer à ceux de la moule zébrée, mieux caractérisés. Dans cette optique, une exposition des deux espèces à un contaminant classique a été réalisée afin de comparer leurs réponses. Les individus ont été exposés à une concentration de 100μg/L de cadmium pendant 7 jours, puis les protéines des branchies ont été séparées par électrophorèse bidimensionnelle et les protéoformes d'abondance variable ont été identifiées par spectrométrie de masse. L'analyse fonctionnelle révèle des éléments prometteurs pour l'étude de la réponse adaptative chez les deux espèces. Une différence d'expression d'acteurs des voies du métabolisme énergétique pointe une différence physiologique, avec une réallocation de l'énergie ainsi que l'apparition de protéines tronquées