NOD2 is ubiquitinated.

<p>A) To determine NOD1 and NOD2 ubiquitination, denatured lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation. Immunoprecipitates were immunoblotted using the indicated antibodies B) To determine the type of ubiquitin-linkage, lysates of HEK293T cells expressing the NOD1 or NOD2 were subjected to immunoprecipitation. Endogenous ubiquitin was revealed using a K48-link specific antibody. C) HEK293T cells were transfected for 72 h with siCTR, siTRIM27-1 or -3 after expression of Flag-NOD2 and HA-Ubiquitin. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) TRIM27 and a E3 mutant of TRIM27 were expressed together with NOD2 and HA-ubiquitin in HEK293T cells. Immunoprecipitates were probed with the indicated antibodies. E) HEK293T cells expressing the indicated proteins were stimulated with 10 µM MDP for the indicated time. Lysates were subjected to immunoprecipitation as described in (C). Densitometric analysis of the TRIM27 signal in the immunoprecipitation normalized to the input signal is shown (bottom). Representative data of at least three independent experiments are shown.</p

TRIM27 contributes to proteasomal degradation of NOD2.

<p>A) HEK293T cells transfected with low amounts of Flag-NOD2 and myc-TRIM27, E3 or CTR as indicated were treated with 30 µg/ml cycloheximid (CHX) and immunoblots of total cell lysates (top) were performed using the indicated antibodies. GAPDH served as loading control. B) HEK293T cells expressing the indicated proteins were treated with 100 nM bortezomib. Lysates were subjected to immunoprecipitation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041255#pone-0041255-g001" target="_blank">Figure 1A</a>. Actin served as loading control. C) HEK293T cells transfected for 48 h with siCTR, siTRIM27-1 or -3 and subsequently with Flag-NOD2 were treated with 30 µg/ml CHX, as indicated. Immunoblots of total cell lysates were performed using the indicated antibodies. GAPDH served as loading control. Representative data of at least three independent experiments are shown (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041255#pone.0041255.s003" target="_blank">Fig. S3B</a>).</p

TRIM27 negatively regulates NOD2 signaling.

<p>A–C) NF-κB luciferase assays in HEK293T cells to determine the influence of TRIM27 overexpression on MDP-induced NOD2-mediated (A), TNF- (B), or IKK-β-induced (C) NF-κB activation. Normalized luciferase activity (nRLU) of unstimulated (white bars) and stimulated (black bars) samples is shown. Values are given as mean+SD (n = 3). *, P<0.05; ***, P<0.005. <i>D.</i> Sections from human colon derived from healthy and active Crohn's disease patients. Staining with DAPI (blue), phalloidin-FITC (green) and with α-TRIM27 antibody (red) is shown. Staining with a rat serum is shown as control in the lower panel (rat serum). Slides are representative for 3 control and 4 Crohn's disease patients. Scale bar = 20 µm. <i>E.</i> qRT-PCR of TRIM27 mRNA expression in colonic biopsies derived from patients with Crohn's disease and controls (CTR) with no evidence of mucosal inflammation. Each symbol represents one patient. TRIM27 mRNA expression normalized to GAPDH is shown. *, P<0.05 (n = 6).</p

NOD2 physically interacts with TRIM27 via the NBD domain.

<p>A–C) Lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation using anti-Flag (A and C) or anti-myc beads (B). Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) Immunoprecipitation of endogenous NOD2 from SW480 cells using the NOD2- specific monoclonal antibody 6F6. Cells were treated with 10 µM MDP for 3 h as indicated. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. E) Protein-protein binding assays using <i>in vitro</i> transcribed and translated [35S]-methionine-labeled NOD2 and recombinant GST or GST-TRIM27 (WT, ΔRING or ΔRING+B-Box) bound to glutathione-Sepharose beads. The coomassie-stained gel (bottom) and the autoradiograph (top) of co-precipitated NOD2 are shown.</p

Mapping of the interaction domains in NOD2 and TRIM27.

<p>The depicted NOD2 (A) or TRIM27 (B) deletion constructs (upper panels) were used for the domain mapping. Lysates were subjected to immunoprecipitation using anti-Flag beads and immunoblotted with the indicated antibodies.</p

Data-sharing in NeurOmics: enabling effective collaboration and working with RD-Connect

<p>Within the NeurOmics project 1100 samples from across 10 rare neurodegenerative and neuromuscular diseases will undergo whole exome sequencing. In addition patients will be deep phenotyped, RNAseq will be carried out and biomarker studies are to be developed – this will all lead to improved understanding of the conditions, causative and modifier gene discovery, more diagnoses and the identification of potential therapeutic targets.</p> <p>For these ambitious aims to be realised, both omics and phenotypic data should be accessible for study by NeurOmics partners across the disease groups. In order to enable this, the NeurOmics project has built an online clinical database in which all phenotypic data is mapped to the Human Phenotype Ontology (HPO), and has established data sharing policies and procedures in close collaboration with RD-Connect. This means that partners are committed to collaborative working within the consortium now and to wider data-sharing via the RD-Connect platform and the European Genome-phenome Archive (EGA) in future, according to agreed timelines that ensure all NeurOmics data is ultimately accessible to researchers worldwide. The policies in place ensure that investigators’ rights to publish first and to intellectual property are protected whilst information sharing is facilitated. They also ensure that NeurOmics complies with the policies of the International Rare Diseases Research Consortium (IRDiRC), which mandates timely sharing of source data for the benefit of the rare disease research community. The NeurOmics policies also recognise the importance of patient consent - where this does not permit wide sharing of anonymised data, patients are to be reconsented before inclusion in the database.</p> <p>These policies have now been agreed and approved by the NeurOmics Steering Committee. This poster outlines how this has been achieved and describes the plans for future working with RD-Connect.</p

SSH1 interacts with NOD1 and regulates cofilin phosphorylation after NOD1 activation.

<p>(A) Co-immunoprecipitation of ectopically expressed SSH1 with NOD1 and NOD2 in human HEK293T cells. Flag-SSH1 was precipitated using a Flag-specific antibody in all samples. (B) Co-immunoprecipitation kinetic of NOD1 and SSH1 in HEK293T cells treated for the indicated time with 500 nM TriDAP. Flag-SSH1 was precipitated using a Flag-specific antibody. (C) <i>In situ</i> PLA detection of the interaction between GFP-SSH1 (shown in green) and Flag-NOD1 in transiently transfected HeLa cells. Protein–protein interactions are visualized as small, distinct red spots (PLA signals). F-Actin (blue) was stained with Alexa633-labeled phalloidin. Shown is a single z section. Scale bar 5 µm. Right panel: Quantification of the total spot area per cell. Significance was calculated by student's t-test (unpaired, two-tailed), p<0.0001. (D) Indirect immunofluorescence study of ectopically expressed myc-SSH1 in HeLa cells after 30 min of infection with <i>S. flexneri</i> (MOI = 10). Bacteria were stained with a LPS specific antibody and are shown in red in the merge image together with DNA staining in blue. Bar: 10 µm. (E) HEK293T cells expressing low amounts of NOD1 were stimulated with TriDAP. IL-8 release (bars) was measured by ELISA (accumulation in supernatant since t = 0) and IL-8 mRNA (line) by qPCR (upper panel). Values are mean + S.D. (n = 3). Cofilin phosphorylation (S3) was monitored by Western Blot analysis (lower panels). Probing for total cofilin and actin served as loading controls. One representative experiment out of three is shown. (F) p-cofilin and total cofilin protein levels were analyzed in HEK293T cells at the indicated time-points after stimulation with 50 ng/ml TNF (upper panel). Alternatively NOD1 was silenced by siRNA treatment for 48 h and cells were stimulated with TriDAP (lower panels) (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s006" target="_blank">Figure S6</a>).</p

NOD1 signaling is dependent on F-actin.

<p>(A) Effect of 6 h cytochalasin D (cytoD) treatment on TriDAP/NOD1 induced NF-κB activation in PMA differentiated THP1-blue reporter cells. Mean + S.D. (n = 3). Significance was calculated by student's t-test (unpaired, two-tailed) *p<0.05, **p<0.005 (compared to 0 nM cytoD). (B) IL-8 secretion of THP1 cells treated for 6 h with the indicated PAMP in the presence or absence of cytochalasin D. Shown is the mean of triplicates from two independent experiments + S.D. (n = 6). The fold induction of IL-8 by cytoD is depicted below. (C) SSH1 knock-down effect on cytoD enhanced NOD1 signaling. Differentiated THP1 cells were treated with cytoD for 6 h and stimulated with 10 µg/ml TriDAP (black bars) or mock treated (open bars). IL-8 secretion measured in the supernatant after 6 h of incubation is shown, mean + S.D. (n = 3), * p<0.05, ** p<0.005.</p

ROCK activity influences NOD1 signaling.

<p>(A) HEK293T cells were transfected with CTRL or siRNA specific for the indicated target and incubated for 48 h. Subsequently, cells were transfected with the NF-κB luciferase reporter and NOD1 and stimulated with TriDAP (filled bars). After 16 h, cells were lysed, luciferase activation was determined and normalized with the β-galactosidase values (nRLU). Values are mean + S.D. (n = 3). Knock-down of the respective target is shown by immunoblot in the right panel. (B) Effect of the expression of constitutively active (CA) and dominant negative (DN) forms of ROCK1 on NOD1-mediated NF-κB activity measured by luciferase reporter assays in HEK293T cells. Values are mean + S.D. (n = 3). (C) NOD1-induced NF-κB activity measured by luciferase reporter assays in HEK293T cells for 6 h with the ROCK inhibitors Y-27632 and Glycyl-H1152. Values are mean + S.D. (n = 3). (D) THP1 cells were treated with the indicated amount of ROCK inhibitor Glycyl-H1152 for 6 h and TriDAP-induced IL-8 secretion was determined by ELISA. Mean + S.D. (n = 3) is shown. Cells were lysed and p-cofilin was detected by Western Blot analysis using a specific antibody (lower panel). Detection of GAPDH served as loading control. (E) Same experimental setting as (D), using HeLa cells stimulated with 10 µg/ml TriDAP. Mean + S.D. is shown. * p<0.05, ** p>0.005 compared to Ctrl (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s008" target="_blank">Figure S8</a>).</p

SSH1 is essential and specific for NOD1-mediated signaling.

<p>(A–B) PMA differentiated THP1-blue cells were treated for 72 h with a non-targeting (siCTRL) or two SSH1-specific siRNA duplex and incubated with TriDAP (10 µg/ml), MDP (10 µg/ml), TNF (0.1 µg/ml), or LPS (0.05 µg/ml). (A) NF-κB activation was measured by SEAP secretion. (B) IL-8 levels in the culture supernatants of the cells from (A). Values are mean + S.D. from two independent experiments conducted in triplicates. Significance was calculated by student's t-test (unpaired, two-tailed) *p<0.05, **p<0.005. n.s.: not significant. (C) Immunoblot of one of the experiments from (A), probing for SSH1 and actin as loading control is shown. (D) Early effects of the knock-down of SSH1. IL-8 (left panel) and SSH1 (right panel) mRNA levels in THP1-blue cells treated as inducted were measured by qPCR. Mean + S.D. from triplicate measurements of one representative experiment is shown (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004351#ppat.1004351.s004" target="_blank">Figure S4</a>).</p