Novel NLRP3/cryopyrin mutations and pro-inflammatory cytokine profiles in Behçet's syndrome patients

The role of mutations in NLRP3 in inflammatory features of Behçet's syndrom

Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease(1). Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways(2,3). Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1 beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1 beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappa B and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1 beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62569/1/nature04517.pd

Editorial "Stem cells: Biology, genetics, and epigenetics"

WOS: 000388500800001Gene therapy, stem cells, and most recently genome editing via CRISPR/CAS9 are among the terms/ methodology that continue to ignite the imagination of all biologists. They hold the potential to overcome previously incurable diseases or even pave the way to genetically designed generations. Although advances in these fields continue at a staggering pace and it seems that we have arrived at our destination, it is sometimes hard to distinguish fiction from reality

A putative role for human BFK in DNA damage-induced apoptosis

Human BFK (BCL-2 family kin) is a novel pro-apoptotic BCL-2 family member specifically expressed in the gastrointestinal tract. BFK has the characteristic BH3 domain, which was shown to be essential for the apoptosis-inducing activity of pro-apoptotic BCL-2 family members. When overexpressed, BFK interacts with BCL-XL and BCL-W but not BCL-2 or BAD in co-immunoprecipitations studies. We find that BFK exhibits striking similarity to BID in the way it is activated through cleavage during apoptosis. The endogenous and cleaved versions of BFK are readily recognized by the rabbit and mouse sera raised against human BFK. An ideal caspase 3 or 7 target sequence, DEVD (amino acids 38–41), is evident N-terminal to the BH3 domain. A recombinant version of the protein containing all residues downstream of the putative caspase cleavage site induces apoptosis in human colon cancer cells, HCT116, and in wild-type mouse embryonic fibroblasts (MEFs), which can be reversed by co-expression of BCL-XL or BCL-W. BFK becomes activated through caspase-dependent cleavage during DNA damage-induced apoptosis. The cleaved form of the protein is dependent on the presence of BAX or BAK for its ability to induce apoptosis, since BAX –/– -BAK –/– double-knockout MEFs are completely resistant to BFK-induced apoptosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63538/1/1046_ftp.pd

Defining Characteristics of Types I and II Apoptotic Cells in Response to TRAIL

Type I cells have been defined to be independent of mitochondria for the induction of Fas death receptor-mediated apoptosis, whereas Type II cells are mitochondria-dependent. Knock-out studies in mice show that thymocytes are Type I and liver cells are Type II. We have previously shown that primary human hepatocytes and HCT116 human colon carcinoma cells behave like Type II cells because TRAIL-induced apoptosis can be blocked by the caspase 9 inhibitor, Z-LEHD-FMK. On the other hand, caspase 9 inhibition does not allow survival of TRAIL-treated SW480 colon cancer cells, which is predicted for Type I cells. Investigating the differences in TRAIL-induced apoptotic pathways in HCT116 and SW480 cells revealed that although FADD, BID, and procaspase 3 protein levels are higher in SW480 cells, and although procaspase 8 and FLIP processing is more efficient at the TRAIL-DISC of SW480 cells, BID, procaspase 3, XIAP, and PARP cleavages occur more rapidly in HCT116, despite the higher levels of BCL-2 and HSP70. Cytochrome c release from the mitochondria to the cytoplasm is more efficient in HCT116 cells. These results suggest BID cleavage as a possible limiting factor in the involvement of mitochondria in TRAIL-induced cell death. Thus, regulation of BID cleavage may define if a cell is mitochondria-dependent or-independent in response to TRAIL death receptor-induced apoptosis

Artificial Loading of ASC Specks with Cytosolic Antigens

<div><p>Inflammasome complexes form upon interaction of Nod Like Receptor (NLR) proteins with pathogen associated molecular patterns (PAPMS) inside the cytosol. Stimulation of a subset of inflammasome receptors including NLRP3, NLRC4 and AIM2 triggers formation of the micrometer-sized spherical supramolecular complex called the ASC speck. The ASC speck is thought to be the platform of inflammasome activity, but the reason why a supramolecular complex is preferred against oligomeric platforms remains elusive. We observed that a set of cytosolic proteins, including the model antigen ovalbumin, tend to co-aggregate on the ASC speck. We suggest that co-aggregation of antigenic proteins on the ASC speck during intracellular infection might be instrumental in antigen presentation.</p></div

Extracellular ASC specks were released from THP-1 macrophages treated with MSU crystals, and can be engulfed and processed by another macrophage.

<p>(A) Human monocytic THP-1 cells were transduced with lentiviral particles to stably express EGFP-ASC fusion protein. THP-1 cells were differentiated with PMA. Differentiated cells were treated with 150 μg/ml MSU crystals for 24 h. ASC speck were observed in the extracellular space (marked with arrows). Bright field is converted into red channel in the overlay image for better visual seperation. Scale bar (A): 100 μm. (B) EGFP-tagged ASC specks were purified from HEK293T cells and incubated with PMA-differentiated THP-1 cells for 3 h. Presence of engulfed ASC speck in an acidic organelle in THP-1 macrophage was demonstrated by Lysotracker Red staining. (C) Time-lapse imaging of PMA-differentiated stably EGFP-ASC expressing THP-1 macrophages that engulfed extracellular mCherry-tagged ASC specks. Tubular vesicles trafficking from the phagolysosome which contained engulfed ASC speck were observed. Cytosolic stable EGFP-ASC expression (diffused green signal) and engulfed mCherry-ASC speck were observed as two distinct compartments within the same cell. Time-lapse imaging was carried out using an in-house produced growth chamber. Results are representative of at least two independent experiments. Scale bar (B) and (C): 25 μm.</p

ASC specks stably co-aggregate some but not all cytosolic proteins.

<p>Fluorescently tagged (mCherry) ASC protein was co-expressed with a set of EGFP-tagged constructs in HEK293T cells. Representative constructs: (A) EGFP-C3 and EGFP alone (B) cOVA-EYFP (1-48aa secretion signal deleted) and EYFP alone were co-expressed with mCherry-ASC. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134912#pone.0134912.s001" target="_blank">S1 Fig</a> for other constructs tested. (C) Shorter versions of C3 and peptide 1 were cloned to C-terminus of EGFP. Only EGFP-peptide 1_19aa co-aggregated on ASC specks, but EGFP-C3_19aa, -peptide 1_12aa and-peptide 1_8aa did not. (D) 2 hydrophobic and 2 hydrophilic randomly generated peptide encoding sequences were cloned to C-terminus of EGFP. EGFP-hydrophobic peptides but not EGFP-hydrophilic peptides co-aggregated on ASC specks. Results are representative of two independent experiments. (E) Hydropathy plots of peptides co-aggregating (peptide 1 and C3) or not co-aggregating (peptide 2 and 3) on ASC specks when fused with EGFP and co-expressed with mCherry-ASC construct. Y-axis: Hydrophobicity values according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134912#pone.0134912.ref038" target="_blank">38</a>]. X-axis: Amino acid position. Columns above the x-axis correspond to hydrophilic peptides and below the x-axis correspond to hydrophobic peptides. (F) EGFP-C3 co-aggregated ASC specks were extracted from HEK293T cells. Samples were imaged right after extraction and after incubation at 37°C for 30 days in PBS. Results are representative of at least two independent experiments. Scale bar: 10 μm.</p

Co-localization frequency of ovalbumin on NLRP3-induced ASC specks is increased by proteasomal inhibition.

<p>HEK293T cells were transduced with lentiviral particles to stably express mCherry-ASC fusion protein. Inverted fluorescent microscopy images of cells, transfected either with empty (pcDNA3) or NLRP3-encoding plasmids, to induce ASC speck formation. Cells were also co-transfected with either (A) EYFP alone (control) or (B) cOVA-EYFP Scale bar (A) and (B): 100 μm. 24 h after transfection, cells were treated with either mock or 10 μM MG132 for 6 h. Exposure time of EYFP alone images is kept 4x shorter than cOVA-EYFP to avoid saturation of pixels due to fluorescence intensity differences. Close-up confocal micrographs of (C) EYFP or (D) cOVA-EYFP expressing NLRP3-induced ASC speck forming cells. Scale bar (C) and (D): 10 μm (E) Western blotting analyses of samples in A-B. EYFP and cOVA-EYFP were exposed equally (marked with asterix). cOVA-EYFP was also exposed longer due to low intensity of the bands. (F) Co-localization frequency of ovalbumin on NLRP3-induced ASC specks either in the absence or presence of MG132 (p < 0.0001, n = 4). (G) NLRP3-induced ASC specks per visual field either in the absence or presence of MG132 (not significant, p = 0.48, n = 4). Results are representative of two independent experiments.</p

A possible founder mutation in FZD6 gene in a Turkish family with autosomal recessive nail dysplasia

Abstract Background Autosomal recessive nail dysplasia is characterized by thick and hard nails with a very slow growth on the hands and feet. Mutations in FZD6 gene were found to be associated with autosomal recessive nail dysplasia in 2011. Presently, only seven mutations have been reported in FZD6 gene; five mutations are clustered in the C-terminus, one is at the seventh transmembrane domain, and another is at the very beginning of third extracellular loop. Methods Whole exome sequencing (WES) was applied to the index case, her one affected sister and her healthy consanguineous parents. The mutation was verified via Sanger sequencing. Molecular dynamics simulations of the predicted structures of native and mutant proteins were compared to gain insight into the pathogenicity mechanism of the mutation. Results Here, we report a homozygous 8 bp deletion mutation, p.Gly559Aspfs*16; c.1676_1683delGAACCAGC, in FZD6 gene which causes a frameshift and creates a premature stop codon at position 16 of the new reading frame. Our molecular dynamics calculations predict that the pathogenicity of this frameshift mutation may be caused by the change in entropy of the protein with negative manner, disturbing the C-terminal domain structure, and hence interaction partners of FZD6. Conclusion We identified a homozygous deletion mutation in FZD6 in a consanguineous Turkish family with nail dysplasia. We also provide a molecular mechanism about the effects of the deletion on the protein structure and its possible motions. This study provides a pathogenicity mechanism for this mutation in nail dysplasia for the first time