RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73

Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the β-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP–TEAD and β-catenin–TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/β-catenin–TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP–p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional “switch” between pluripotency and initiation of differentiation

Granulin Is a Soluble Cofactor for Toll-like Receptor 9 Signaling

Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling.National Institutes of Health (U.S.)Novartis (Firm

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family

Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo

Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes

Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery

Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2′-C-methylcytidine (2′CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies

ASPP2 Links the Apical Lateral Polarity Complex to the Regulation of YAP Activity in Epithelial Cells

<div><p>The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP <i>in vitro</i>, in epithelial cells, and <i>in vivo</i>, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.</p></div

ASPP2 scaffolds PP1 to dephosphorylate YAP.

<p>(<b>A</b>) The importance of the ASPP2/PP1 interaction in dephosphorylating YAP was tested by transfecting the indicated constructs in HEK293 cells. Exogenous YAP was subsequently immunoprecipitated using an anti-myc mouse monoclonal antibody (9E10) and SDS-Page/immunoblotting was performed using the indicated antibodies. The bar graph represents the ratio between pYAP S127 and total YAP protein levels (n = 3; *: p<0.05; n.s.: non-significant). Error bars indicate standard deviation. (<b>B</b>) Lysates obtained from HEK293 cells transfected with the indicated constructs were analysed by SDS-PAGE/immunoblotting on a Phos-Tag gel. (<b>C</b>) The ability of ASPP2 (Y869A/Y874A)-V5 to dephosphorylate YAP was tested in HEK293 cells to investigate the importance of the YAP/ASPP2 interaction in this process. Following transfection with the indicated constructs in HEK293 cells, Exogenous YAP was immunoprecipitated using an anti-myc mouse monoclonal antibody (9E10) and SDS-Page/immunoblotting was performed using the indicated antibodies. (<b>D</b>) The ability of ASPP2, iASPP and ASPP1-V5 to interact with and dephosphorylate YAP was tested in HEK293 cells. Following transfection with the indicated constructs in HEK293 cells, Exogenous YAP was immunoprecipitated using an anti-myc mouse monoclonal antibody (9E10) and SDS-Page/immunoblotting was performed using the indicated antibodies.</p

ASPP2 promotes the transcriptional activity of YAP.

<p>(<b>A</b>) Following transfection of Caco-2 cells with control or ASPP2 siRNA, the localisation of YAP and ASPP2 was analysed by immunostaining at low and high cell density. Scale bar: 20 µm. (<b>B</b>) The ability of wild type ASPP2 and ASPP2 (R<u>A</u>K<u>A</u>)-V5 to regulate TEAD-mediated transcription was analysed in a luciferase assay using the TEAD-luciferase reporter (8xGTIIC-luciferase) in Caco-2 cells. Values were obtained from three independent duplicate experiments and error bars indicate standard deviation (*: p<0.05). (<b>C</b>) YAP immunostaining of paraffin sections obtained from the colons of wild type and <i>ASPP2</i>^Δexon3 mice. Dashed white squares highlight magnified areas represented in the corresponding panels (C′-C′″). White arrowheads point to YAP positive nuclei in wild type crypts whereas yellow arrowheads point to nuclei devoid of YAP in <i>ASPP2</i>^Δexon3 crypts. Nuclei were counterstained with DAPI. Scale bars: 50 µm. (<b>D</b>) <i>CTGF</i> mRNA levels were quantified by qRT-PCR using RNA obtained from the colons of wild type (n = 3) and <i>ASPP2</i>^Δexon3 mice (n = 3). Error bars represent standard deviation (*: p<0.05). (<b>E</b>) Diagram representing the regulation of YAP by ASPP2 in epithelial cells. Once YAP is phosphorylated at serine 127, it can interact with ASPP2 at the apical lateral domain where ASPP2 induces its dephosphorylation via the recruitment of PP1. YAP is consequently able to relocalise to the nucleus where it can modulate TEAD transcriptional activity.</p

Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis

Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes