Computational and experimental analysis of bioactive peptide linear motifs in the integrin adhesome

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation

Pregnancy-specific glycoproteins bind integrin alpha IIb beta 3 and inhibit the platelet-fibrinogen interaction

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 mu g/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGF beta 1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin alpha IIb beta 3 antagonist found in snake venom, suggested that PSG1 may be a selective alpha IIb beta 3 ligand. Here we show that human PSG1 binds alpha IIb beta 3 and inhibits the platelet - fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit alpha IIb beta 3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml) fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy

Human and mouse PSGs inhibit the platelet – fibrinogen interaction.

<p>PSG-mediated inhibition of the platelet – fibrinogen interaction was measured by estimating binding of Oregon Green-conjugated fibrinogen (OgFg) to washed human platelets using FACS. Fibrinogen binding to TRAP-activated platelets is set at 100% and resting platelets at 0%. All assays were analysed over a four or five point dose range of PSG proteins and mutants, from ∼5–100 or 200 µg/ml, depending on protein molecular weight. For clarity, some results are reported as single dose molar concentration comparisons between proteins. Protein molecular weights were calculated from amino acid sequences with no adjustments for posttranslational modifications. <b>a,</b> Binding of OgFg to human platelets in the presence of human CEACAM1, human IgG, and increasing doses of recombinant wildtype human PSG1. 4 µM PSG1 is equivalent to 200 µg/ml protein. <b>b,</b> Binding of OgFg to human platelets in the presence of (left to right): wildtype PSG1 (KGD); PSG1 in which the KGD tri-peptide motif is replaced with RGE, or AAA; PSG1 with deletion of N-domain; PSG1 N-domain; PSG1 N-domain in which the KGD tri-peptide motif is replaced with AAA. All proteins were used at 2 µM concentration, equivalent to 100 µg/ml full-length PSG1 variants, 75 µg/ml for PSG1ΔN, and 38 µg/ml for PSG1N variants. <b>c</b> & <b>d,</b> Binding of OgFg to human platelets in the presence of increasing concentrations of recombinant human PSG9 and mouse Psg23, respectively. 2 µM PSG9 and 2 µM Psg23 is equivalent to 100 µg/ml and 110 µg/ml, respectively. <b>e,</b> Summary of domain structures and mutants of PSG proteins used (see Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057491#pone.0057491.s001" target="_blank">File S1</a> for sequences). <b>f,</b> Representative Coomassie-stained gels of protein used. For a - d, data are means of between three and seven independent experiments (detailed in main text) ± S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001, nonparametric ANOVA with Dunnett’s multiple comparison post test.</p

Multiple domains of human PSG1 bind the platelet integrin αIIbβ3.

<p><b>a,</b> Integrin αIIbβ3 (2µg purified protein; lanes 1–3) pulls down PSG1 in an <i>in vitro</i> binding assay (lane 1). Negative controls are Protein G agarose beads with (lane 2) and without (lane 4) αIIbβ3, and with rabbit IgG instead of PSG1 (lane 3). Similarly, αIIbβ3 from lysates of CHO cell line stably transfected with αIIbβ3 (lanes 7, 8), but not lysate of sham transfected CHO control cell line (lanes 5, 6) pulls down PSG1 in co-immunoprecipitation assays. Negative controls lack PSG1, but contain α-αIIbβ3 mAb bound to beads (lanes 5 & 7). Western blotted membranes were probed with α-αIIbβ3 mAb Sz22 (upper gel) and α-PSG1 mAb-5 (lower gel). <b>b,</b> Commercial purified integrin αIIbβ3 bound to Protein G agarose beads pulls down recombinant PSG1 (lane 1) and PSG1ΔN (lane 2). Negative controls lack PSG1 (lane 3) or αIIbβ3 (lane 4). Western blotted membranes were probed with α-αIIbβ3 mAb Sz22 (upper gel), and α-His-Tag pAb (lower gel) which detects tagged PSG1 and PSG1ΔN proteins. <b>c</b>, Representative image and pooled data of fluorescent PSG1 (PSG1–800) binding to CHO cell line stably transfected with αIIbβ3 compared to sham transfected CHO control cell line. Cell density was measured using SYTO60. Data are means of six independent experiments ± S.E.M. *, P<0.05, Paired Student’s t-test. <b>d,</b> Binding of the activation-dependent monoclonal antibody, PAC-1, to platelet αIIbβ3. Washed human platelets were preincubated with BSA or PSG1 at 200 µg/ml before the addition of PAC-1 antibody and the indicated platelet agonist: TRAP (4 µM), thromboxane mimetic U46619 (250 nM), ADP (10 µM) or epinephrine (25 µM). Data are means of four independent experiments ± S.E.M. *, P<0.05, Student’s t-test. <b>e,</b> Washed platelets adhere and spread extensively on fibrinogen-coated (20 µg/ml) glass slides but poorly on 1% BSA-coated slides. Pre-incubation of platelets with 200 µg/ml PSG1 significantly reduced platelet adhesion and spreading on fibrinogen. Permeabilized platelets were stained for polymerized F-actin with Alexa-488 fluorescein isothiocyanate phalloidin before visualisation using confocal microscopy. Representative images are shown. Scale bar is 20 µm. Graph shows quantification of platelet adhesion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057491#s4" target="_blank">Methods</a>. Data are means of three independent experiments ± S.E.M. *, P<0.05, Student’s t-test.</p

PSG1 does not activate platelets.

<p><b>a,</b> Washed human platelets were treated at 37°C for 3 min with TRAP (4 µM) and/or PSG1 (200 µg/ml) for 2 min as indicated. Alternatively platelets remained untreated (resting). Platelet activation was assessed by analysis of the phosphotyrosine profile by western blotting with the antiphosphotyrosine mAb 4G10. Experiment was performed twice. <b>b, c</b> & <b>d,</b> In a similar series of experiments, three different markers of platelet degranulation were assessed. For ADP secretion assay (b), platelets were treated with 4 µM TRAP and/or PSG1 (100 µg/ml) for 3 min at 37^OC. For surface expression of CD62P (c) and CD63 (d) platelets were treated with 4 µM TRAP and/or PSG1 (100 µg/ml) for 10 min at RT as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057491#s4" target="_blank">Methods</a>. Alternatively platelets remained untreated (resting). Data represent the means of three independent experiments ± S.E.M.</p