Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR(2)), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR(2) deubiquitination and its importance in trafficking and signaling of endocytosed PAR(2) are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR(2) ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR(2). Trapping PAR(2) in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR(2) with beta-arrestin2 or the duration of PAR(2)-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR(2) but not for regulating PAR(2) dissociation from beta-arrestin2 or PAR(2)-mediated ERK2 activation.

Endothelin-converting enzyme-1 degrades internalized somatostatin-14

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR(2). In HEK-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR(2) and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.

c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR(2)), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR(2) is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR(2) was mono-ubiquitinated. Immunofluorescence indicated that activated PAR(2) translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR(2) lacking intracellular lysine residues (PAR(2)Delta14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR(2) Delta14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR(2) stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR(2) at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR(2) ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR(2) was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR(2) to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR(2) to irrevocably terminate signaling by this and perhaps other GPCRs.

Trypsin IV, a novel agonist of protease-activated receptors 2 and 4

Certain serine proteases signal to cells by cleaving protease-activated receptors (PARs) and thereby regulate hemostasis, inflammation, pain and healing. However, in many tissues the proteases that activate PARs are unknown. Although pancreatic trypsin may be a physiological agonist of PAR(2) and PAR(4) in the small intestine and pancreas, these receptors are expressed by cells not normally exposed pancreatic trypsin. We investigated whether extrapancreatic forms of trypsin are PAR agonists. Epithelial cells lines from prostate, colon, and airway and human colonic mucosa expressed mRNA encoding PAR(2), trypsinogen IV, and enteropeptidase, which activates the zymogen. Immunoreactive trypsinogen IV was detected in vesicles in these cells. Trypsinogen IV was cloned from PC-3 cells and expressed in CHO cells, where it was also localized to cytoplasmic vesicles. We expressed trypsinogen IV with an N-terminal Igkappa signal peptide to direct constitutive secretion and allow enzymatic characterization. Treatment of conditioned medium with enteropeptidase reduced the apparent molecular mass of trypsinogen IV from 36 to 30 kDa and generated enzymatic activity, consistent with formation of trypsin IV. In contrast to pancreatic trypsin, trypsin IV was completely resistant to inhibition by polypeptide inhibitors. Exposure of cell lines expressing PAR(2) and PAR(4) to trypsin IV increased [Ca(2+)](i) and strongly desensitized cells to PAR agonists, whereas there were no responses in cells lacking these receptors. Thus, trypsin IV is a potential agonist of PAR(2) and PAR(4) in epithelial tissues where its resistance to endogenous trypsin inhibitors may permit prolonged signaling.

Protease-activated receptors: the role of cell-surface proteolysis in signalling

Certain extracellular proteases, derived from the circulation and inflammatory cells, can specifically cleave and trigger protease-activated receptors (PARs), a small, but important, sub-group of the G-protein-coupled receptor super-family. Four PARs have been cloned and they all share the same basic mechanism of activation: proteases cleave at a specific site within the extracellular N-terminus to expose a new N-terminal tethered ligand domain, which binds to and thereby activates the cleaved receptor. Thrombin activates PAR1, PAR3 and PAR4, trypsin activates PAR2 and PAR4, and mast cell tryptase activates PAR2 in this manner. Activated PARs couple to signalling cascades that affect cell shape, secretion, integrin activation, metabolic responses, transcriptional responses and cell motility. PARs are 'single-use' receptors: proteolytic activation is irreversible and the cleaved receptors are degraded in lysosomes. Thus, PARs play important roles in 'emergency situations', such as trauma and inflammation. The availability of selective agonists and antagonists of protease inhibitors and of genetic models has generated evidence to suggests that proteases and their receptors play important roles in coagulation, inflammation, pain, healing and protection. Therefore, selective antagonists or agonists of these receptors may be useful therapeutic agents for the treatment of human diseases.

Protease-activated receptors: protease signaling in the gastrointestinal tract

Serine proteases from the circulation, inflammatory cells, digestive glands and microorganisms can signal to cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors. Proteases cleave PARs at specific sites to expose tethered ligand domains that bind to and activate the cleaved receptors. Despite this irreversible mechanism of activation, PAR signaling is tightly regulated to prevent the uncontrolled stimulation of cells. Although PARs are found in all organ systems, protease signaling is of particular interest in the gastrointestinal tract, where proteases regulate neurotransmission, secretion, motility, epithelial permeability and intestinal inflammation, and can thus contribute to disease.

The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Site-specific and geographical segmental social, environmental and ethical disclosures by the Ghanaian mining sector

Purpose: This paper explores the extent of site-specific and geographic segmental social, environmental and ethical reporting by mining companies operating in Ghana. We aim to: (i) establish a picture of corporate transparency relating to geographic segmentation of social, environmental and ethical reporting which is specific to operating sites and country of operation, and; (ii) gauge the impact of the introduction of integrated reporting on site-specific social, environmental and ethical reporting. Methodology/Approach: We conducted an interpretive content analysis of the annual/integrated reports of mining companies for the years 2009, 2010 and 2011 in order to extract site-specific social, environmental and ethical information relating to the companies’ mining operations in Ghana. Findings and Implications: We found that site-specific social, environmental and ethical reporting is extremely patchy and inconsistent between the companies’ reports studied. We also found that there was no information relating to certain sites, which were in operation, according to the Ghana Minerals Commission. This could simply be because operations were not in progress. Alternatively it could be that decisions are made concerning which site-specific information is reported according to a certain benchmark. One policy implication arising from this research is that IFRS should require geographic segmental reporting of material social, environmental and ethical information in order to bring IFRS into line with global developments in integrated reporting. Originality: Although there is a wealth of sustainability reporting research and an emergent literature on integrated reporting, there is currently no academic research exploring site-specific social, environmental and ethical reporting

A Lab-in-a-briefcase for rapid prostate specific antigen (PSA) screening from whole blood

We present a new concept for rapid and fully portable Prostate Specific Antigen (PSA) measurement, termed “Lab-in-a-Briefcase”, which integrates an affordable microfluidic ELISA platform utilising a melt-extruded fluoropolymer Micro Capillary Film (MCF) containing 10 bore, 200 μm internal diameter capillaries, a disposable multi-syringe aspirator (MSA) plus a sample tray pre-loaded with all required immunoassay reagents, and a portable film scanner for colorimetric signal digital quantitation. Each MSA can perform 10 replicate microfluidic immunoassays on 8 samples, allowing 80measurements to be made in less than 15 minutes based on semi-automated operation and norequirement of additional fluid handling equipment. An assay was optimised for measurement of a clinically relevant range of PSA from 0.9 to 60.0 ng/ml in 15 minutes with CVs in the order of 5% based on intra-assay variability when read using a consumer flatbed film scanner. The PSA assay performance in the MSA remained robust in the presence of undiluted or 1:2 diluted human serum or whole blood, and the matrix effect could simply be overcome by extending sample incubation times. The PSA "Lab-in-a-briefcase" is particularly suited to a low-resource health setting where diagnostic labs and automated immunoassay systems are not accessible, by allowing PSA measurement outside the laboratory using affordable equipment.

Potential influences on the United Kingdom's floods of winter 2013/14

During the winter of 2013/14, much of the UK experienced repeated intense rainfall events and flooding. This had a considerable impact on property and transport infrastructure. A key question is whether the burning of fossil fuels is changing the frequency of extremes, and if so to what extent. We assess the scale of the winter flooding before reviewing a broad range of Earth system drivers affecting UK rainfall. Some drivers can be potentially disregarded for these specific storms whereas others are likely to have increased their risk of occurrence. We discuss the requirements of hydrological models to transform rainfall into river flows and flooding. To determine any general changing flood risk, we argue that accurate modelling needs to capture evolving understanding of UK rainfall interactions with a broad set of factors. This includes changes to multiscale atmospheric, oceanic, solar and sea-ice features, and land-use and demographics. Ensembles of such model simulations may be needed to build probability distributions of extremes for both pre-industrial and contemporary concentration levels of atmospheric greenhouse gases.

Mode-specificity and transition state-specific energy redistribution in the chemisorption of CH4 on Ni{100}

We have investigated methane (CH4) dissociative chemisorption on the Ni{100} surface by first-principles molecular dynamics (MD) simulations. Our results show that this reaction is mode-specific, with the n1 state being the most strongly coupled to efficient energy flow into the reaction coordinate when the molecule reaches the transition state. By performing MD simulations for two different transition state (TS) structures we provide evidence of TS structure-specific energy redistribution in methane chemisorption. Our results are compared with recently reported state-resolved measurement of methane adsorption probability on nickel surfaces, and we find that a strong correlation exists between the highest vibrational efficacy measured on Ni{100} for the n1 state and the calculated highest fractional vibrational energy content in this mode.

Do location specific forecasts pose a new challenge for communicating uncertainty?

In the last decade, the growth of local, site-specific weather forecasts delivered by mobile phone or website represents arguably the fastest change in forecast consumption since the beginning of Television weather forecasts 60 years ago. In this study, a street-interception survey of 274 members of the public a clear first preference for narrow weather forecasts above traditional broad weather forecasts is shown for the first time, with a clear bias towards this preference for users under 40. The impact of this change on the understanding of forecast probability and intensity information is explored. While the correct interpretation of the statement ‘There is a 30% chance of rain tomorrow’ is still low in the cohort, in common with previous studies, a clear impact of age and educational attainment on understanding is shown, with those under 40 and educated to degree level or above more likely to correctly interpret it. The interpretation of rainfall intensity descriptors (‘Light’, ‘Moderate’, ‘Heavy’) by the cohort is shown to be significantly different to official and expert assessment of the same descriptors and to have large variance amongst the cohort. However, despite these key uncertainties, members of the cohort generally seem to make appropriate decisions about rainfall forecasts. There is some evidence that the decisions made are different depending on the communication format used, and the cohort expressed a clear preference for tabular over graphical weather forecast presentation.