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

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

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

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

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

Recombinant human serum amyloid P in healthy volunteers and patients with pulmonary fibrosis

PRM-151, recombinant human Pentraxin-2 (PTX-2) also referred to as serum amyloid P (SAP), is under development for treatment of fibrosis. A First-in-Human (FIH) trial was performed to assess the safety, tolerability, and pharmacokinetics of single ascending intravenous doses of PRM-151 administered to healthy subjects, using a randomized, blinded, placebo controlled study design. Each cohort included three healthy subjects (PRM-151:placebo; 2:1). SAP levels were assessed using a validated ELISA method, non-discriminating between endogenous and exogenous SAP. At a dose level of 10 mg/kg, at which a physiologic plasma level of SAP was reached, two additional healthy volunteers and three pulmonary fibrosis (PF) patients were enrolled enabling comparison of the pharmacokinetic SAP profile between healthy volunteers and PF patients. In addition, the percentage of fibrocytes (CD45+/Procollagen-1+ cells) in whole blood samples was assessed to demonstrate biological activity of PRM-151 in the target population. PRM-151 administration was generally well tolerated. In two pulmonary fibrosis patients non-specific, transient skin reactions (urticaria and erythema) were observed. PRM-151 administration resulted in a 6- to 13-fold increase in mean baseline plasma SAP levels at dose levels of 5, 10, and 20 mg/kg. The estimated t(1/2) of PRM-151 in healthy volunteers was 30 h. Pharmacokinetic profiles were comparable between healthy volunteers and PF patients. PRM-151 administration resulted in a 30-50% decrease in fibrocyte numbers 24 h post-dose. This suggests that administration of PRM-151 may be associated with a reduction of fibrocytes in PF patients, a population for which current pharmacotherapeutic options are limited. The pharmacological action of PRM-151 should be confirmed in future research. (C) 2013 Elsevier Ltd. All rights reserved

Structural basis for a novel intrapeptidyl H-bond and reverse binding of c-Cbl-TKB domain substrates

The c-Cbl tyrosine kinase binding domain (Cbl-TKB), essentially an ‘embedded' SH2 domain, has a critical role in targeting proteins for ubiquitination. To address how this domain can bind to disparate recognition mofits and to determine whether this results in variations in substrate-binding affinity, we compared crystal structures of the Cbl-TKB domain complexed with phosphorylated peptides of Sprouty2, Sprouty4, epidermal growth factor receptor, Syk, and c-Met receptors and validated the binding with point-mutational analyses using full-length proteins. An obligatory, intrapeptidyl H-bond between the phosphotyrosine and the conserved asparagine or adjacent arginine is essential for binding and orientates the peptide into a positively charged pocket on c-Cbl. Surprisingly, c-Met bound to Cbl in the reverse direction, which is unprecedented for SH2 domain binding. The necessity of this intrapeptidyl H-bond was confirmed with isothermal titration calorimetry experiments that also showed Sprouty2 to have the highest binding affinity to c-Cbl; this may enable the selective sequestration of c-Cbl from other target proteins