Regulation of early steps of GPVI signal transduction by phosphatases: a systems biology approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation

Calcium Flux in Neutrophils Synchronizes β2 Integrin Adhesive and Signaling Events that Guide Inflammatory Recruitment

Intracellular calcium flux is an early step in the signaling cascade that bridges ligation of selectin and chemokine receptors to activation of adhesive and motile functions during recruitment on inflamed endothelium. Calcium flux was imaged in real time and provided a means of correlating signaling events in neutrophils rolling on E-selectin and stimulated by chemokine in a microfluidic chamber. Integrin dependent neutrophil arrest was triggered by E-selectin tethering and ligation of IL-8 seconds before a rapid rise in intracellular calcium, which was followed by the onset of pseudopod formation. Calcium flux on rolling neutrophils increased in a shear dependent manner, and served to link integrin adhesion and signaling of cytoskeletally driven cell polarization. Abolishing calcium influx through membrane expressed store operated calcium channels inhibited activation of high affinity β2 integrin and subsequent cell arrest. We conclude that calcium influx at the plasma membrane integrates chemotactic and adhesive signals, and functions to synchronize signaling of neutrophil arrest and migration in a shear stress dependent manner

Additional Serine/Threonine Phosphorylation Reduces Binding Affinity but Preserves Interface Topography of Substrate Proteins to the c-Cbl TKB Domain

The E3-ubiquitin ligase, c-Cbl, is a multi-functional scaffolding protein that plays a pivotal role in controlling cell phenotype. As part of the ubiquitination and downregulation process, c-Cbl recognizes targets, such as tyrosine kinases and the Sprouty proteins, by binding to a conserved (NX/R)pY(S/T)XXP motif via its uniquely embedded SH2 domain (TKB domain). We previously outlined the mode of binding between the TKB domain and various substrate peptide motifs, including epidermal growth factor receptor (EGFR) and Sprouty2 (Spry2), and demonstrated that an intrapetidyl hydrogen bond forms between the (pY-1) arginine or (pY-2) asparagine and the phosphorylated tyrosine, which is crucial for binding. Recent reports demonstrated that, under certain types of stimulation, the serine/threonine residues at the pY+1 and/or pY+2 positions within this recognition motif of EGFR and Sprouty2 may be endogenously phosphorylated. Using structural and binding studies, we sought to determine whether this additional phosphorylation could affect the binding of the TKB domain to these peptides and consequently, whether the type of stimulation can dictate the degree to which substrates bind to c-Cbl. Here, we show that additional phosphorylation significantly reduces the binding affinity between the TKB domain and its target proteins, EGFR and Sprouty2, as compared to peptides bearing a single tyrosine phosphorylation. The crystal structure indicates that this is accomplished with minimal changes to the essential intrapeptidyl bond and that the reduced strength of the interaction is due to the charge repulsion between c-Cbl and the additional phosphate group. This obvious reduction in binding affinity, however, indicates that Cbl's interactions with its TKB-centered binding partners may be more favorable in the absence of Ser/Thr phosphorylation, which is stimulation and context specific in vivo. These results demonstrate the importance of understanding the environment in which certain residues are phosphorylated, and the necessity of including this in structural investigations

Alterations in Adenosine Metabolism and Signaling in Patients with Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis

Background: Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an upregulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated. Methodology/Principal Findings: The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A2BR are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A2BR in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A 2BR, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A 2BR on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators. Conclusions/Significance: These findings suggest that components of adenosine metabolism and signaling are altered in

Identifying the Rules of Engagement Enabling Leukocyte Rolling, Activation, and Adhesion

The LFA-1 integrin plays a pivotal role in sustained leukocyte adhesion to the endothelial surface, which is a precondition for leukocyte recruitment into inflammation sites. Strong correlative evidence implicates LFA-1 clustering as being essential for sustained adhesion, and it may also facilitate rebinding events with its ligand ICAM-1. We cannot challenge those hypotheses directly because it is infeasible to measure either process during leukocyte adhesion following rolling. The alternative approach undertaken was to challenge the hypothesized mechanisms by experimenting on validated, working counterparts: simulations in which diffusible, LFA1 objects on the surfaces of quasi-autonomous leukocytes interact with simulated, diffusible, ICAM1 objects on endothelial surfaces during simulated adhesion following rolling. We used object-oriented, agent-based methods to build and execute multi-level, multi-attribute analogues of leukocytes and endothelial surfaces. Validation was achieved across different experimental conditions, in vitro, ex vivo, and in vivo, at both the individual cell and population levels. Because those mechanisms exhibit all of the characteristics of biological mechanisms, they can stand as a concrete, working theory about detailed events occurring at the leukocyte–surface interface during leukocyte rolling and adhesion experiments. We challenged mechanistic hypotheses by conducting experiments in which the consequences of multiple mechanistic events were tracked. We quantified rebinding events between individual components under different conditions, and the role of LFA1 clustering in sustaining leukocyte–surface adhesion and in improving adhesion efficiency. Early during simulations ICAM1 rebinding (to LFA1) but not LFA1 rebinding (to ICAM1) was enhanced by clustering. Later, clustering caused both types of rebinding events to increase. We discovered that clustering was not necessary to achieve adhesion as long as LFA1 and ICAM1 object densities were above a critical level. Importantly, at low densities LFA1 clustering enabled improved efficiency: adhesion exhibited measurable, cell level positive cooperativity

Expression and genomic analysis of midasin, a novel and highly conserved AAA protein distantly related to dynein

BACKGROUND: The largest open reading frame in the Saccharomyces genome encodes midasin (MDN1p, YLR106p), an AAA ATPase of 560 kDa that is essential for cell viability. Orthologs of midasin have been identified in the genome projects for Drosophila, Arabidopsis, and Schizosaccharomyces pombe. RESULTS: Midasin is present as a single-copy gene encoding a well-conserved protein of ~600 kDa in all eukaryotes for which data are available. In humans, the gene maps to 6q15 and encodes a predicted protein of 5596 residues (632 kDa). Sequence alignments of midasin from humans, yeast, Giardia and Encephalitozoon indicate that its domain structure comprises an N-terminal domain (35 kDa), followed by an AAA domain containing six tandem AAA protomers (~30 kDa each), a linker domain (260 kDa), an acidic domain (~70 kDa) containing 35–40% aspartate and glutamate, and a carboxy-terminal M-domain (30 kDa) that possesses MIDAS sequence motifs and is homologous to the I-domain of integrins. Expression of hemagglutamin-tagged midasin in yeast demonstrates a polypeptide of the anticipated size that is localized principally in the nucleus. CONCLUSIONS: The highly conserved structure of midasin in eukaryotes, taken in conjunction with its nuclear localization in yeast, suggests that midasin may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus. The AAA domain of midasin is evolutionarily related to that of dynein, but it appears to lack a microtubule-binding site

Impact of Evidence-Based Interventions on Nurse Burnout Among Ambulatory Care Nurses

Background: The physical and emotional demands of the nursing profession often cause increased stress and burnout in nurses. If burnout is not addressed, the negative consequences of burnout are perpetuated by one another. Evidence-based interventions, such as meditation, yoga, and physical activity can help reduce nurse burnout. Local Problem: This project was completed at an ambulatory dermatology clinic that sees approximately 300 patients a day. In recent years, this department has noticed increased nurse turnover and low nurse morale. Methods: A convenience sample from approximately 30 nurses was utilized. Burnout was measured using the Maslach Burnout Inventory for Medical Personnel pre- and postintervention. Interventions: Every week, participants chose to complete meditation, yoga, or a physical activity within the FitOn smartphone application (app) for 30 minutes 5 days a week. Participants were encouraged to switch between meditation, yoga, and physical activity each day. Results: Seven nurses initially participated in the project, and one participant completed the project in its entirety. The participant’s emotional exhaustion score decreased from 31 out of 54 to 9, depersonalization decreased from 3 out of 30 to 0, and personal accomplishment remained unchanged at 37 out of 48. Conclusion: Additional projects are needed to further examine the impact of meditation, yoga, and physical activity on nurse burnout

Meiofauna mediates ecosystem effects of anthropogenic disturbances in the oceans.

Humans have used, and had effects on, marine ecosystems, particularly during the recent history. As the human population and its economic activities increase, these effects intensify at the global scale and at all depths. Yet, our awareness and understanding of the long‐term, pervasive effects of anthropogenic disturbances on the seafloor, and the resident meiofauna, are far from complete. Here, we explore meiofauna responses to, and their recovery from, the most prominent and widespread anthropogenic disturbances in the sea, including bottom trawling, pollution, the spread of invasive species, and climate change. Anthropogenic disturbance and natural environmental dynamics interact, causing changes in meiofaunal abundance and diversity, either in the short-term, through effects on growth and development, or in the long-term, through genetic selection or adaptation to changing environmental conditions. Species-specific sensitivity to disturbance can propagate to community-level responses, mediated by shifts in interspecific interactions. Meiofaunal responses to anthropogenic disturbance are typically non-linear and depend on the environmental context in which the disturbance occurs, on the spatial and temporal scales of observation, and on the extent to which the disturbance creates novel environments to which the meiofauna has to adapt. Due to their small size, life history characteristics, and phylogenetic and functional diversity, meiofaunal assemblages are highly resilient, and there is little evidence for the local extinction of meiofauna from anthropogenically disturbed seafloor habitats. It therefore seems likely that meiofauna has the ability to adapt, and thrive, in response to most environmental changes. This assumption, however, is being challenged as several environmental elements (e.g., atmospheric CO2 concentrations, biogeochemical cycles, etc.) are moving beyond their historical ranges. Placing meiofauna responses to anthropogenic disturbance in the wider context of benthic ecosystems, we conclude by emphasising current uncertainties regarding observable versus assumed, direct versus indirect, and single versus interactive consequences of anthropogenic disturbances

Hidden Players—Meiofauna Mediate Ecosystem Effects of Anthropogenic Disturbances in the Ocean.

Humans have used, and had effects on, marine ecosystems throughout history. As the human population and its economic activities increase, these effects intensify. Yet, our awareness and understanding of the long‐term, pervasive effects of anthropogenic disturbances on the seafloor, and the resident meiofauna, is far from complete. This chapter summarises research on the responses of marine meiofauna to the most widespread anthropogenic disturbances, including bottom-fishing, pollution, introduction of invasive species, and climate change. Anthropogenic disturbance and natural environmental dynamics interact to cause changes in the response of meiofauna species, either in the short-term, through effects on growth and development, or in the long-term, through genetic selection. Species-specific sensitivity to disturbance can propagate to community-level responses, mediated by shifts in interspecific interactions. Meiofauna responses to anthropogenic disturbance are commonly nonlinear and depend on the environmental context in which the disturbance occurs, on the scales at which meiofauna responses are observed, and on the extent to which the disturbance creates novel environments that differ from those to which the resident meiofauna are adapted. Although responses of meiofauna assemblages to anthropogenic disturbance are complex, in general severe disturbance leads to dominance by opportunistic species. The widespread replacement of habitat-specific ecological specialists by broadly-adapted ecological generalists and opportunists often results in biotic and functional homogenisation of once disparate biotas. Their small size, their life history characteristics, and their phylogenetically and functionally diverse species pool, all suggest that meiofauna are resilient, and there is little evidence for the local extinction of meiofauna from anthropogenically disturbed seafloor habitats. It therefore seems likely that meiofauna have the ability to adapt, and thrive, in response to most environmental changes. New horizons for future meiofauna research pertain to the extent to which the resistance or resilience of meiofauna to anthropogenic disturbance buffers ecosystem functioning against further change