The familial Mediterranean fever protein, pyrin, is cleaved by caspase-1 and activates NF-kappaB through its N-terminal fragment.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in MEFV, which encodes a 781-amino acid protein denoted pyrin. We have previously shown that pyrin regulates caspase-1 activation and IL-1beta production through interaction of its N-terminal PYD motif with the ASC adapter protein, and also modulates IL-1beta production by interaction of its C-terminal B30.2 domain with the catalytic domains of caspase-1. We now asked whether pyrin might itself be a caspase-1 substrate, and found that pyrin is cleaved by caspase-1 at Asp330, a site remote from the B30.2 domain. Pyrin variants harboring FMF-associated B30.2 mutations were cleaved more efficiently than wild-type pyrin. The N-terminal cleaved fragment interacted with the p65 subunit of NF-kappaB and with IkappaB-alpha through its 15-aa bZIP basic domain and adjacent sequences, respectively, and translocated to the nucleus. The interaction of the N-terminal fragment with p65 enhanced entrance of p65 into the nucleus. The interaction of N-terminal pyrin with IkappaB-alpha induced calpain-mediated degradation of IkappaB-alpha, thus potentiating NF-kappaB activation. Absolute and relative quantities of cleaved pyrin and IkappaB-alpha degradation products were substantially increased in leukocytes from FMF patients compared with healthy controls. Our data support a new pyrin/caspase-1 pathway for NF-kappaB activation.

Human mast cells release oncostatin M on contact with activated T cells: possible biologic relevance.

BACKGROUND: We have recently demonstrated that mast cells can be activated by heterotypic adhesion to activated T cells. OBJECTIVE: We sought to perform gene expression profiling on human mast cells activated by either IgE cross-linking or by T cells and to characterize one of the cytokines, oncostatin M (OSM). METHODS: Gene expression profiling was done by means of microarray analysis, OSM expression was validated by means of RT-PCR, and the product was measured by means of ELISA in both the LAD 2 human mast cell line and in cord blood-derived human mast cells. Immunocytochemistry was used to localize OSM in human mast cells, and its biologic activity was verified by its effect on the proliferation of human lung fibroblasts. RESULTS: OSM was expressed and released specifically on T cell-induced mast cell activation but not on IgE cross-linking. OSM was localized to the cytoplasm, and its expression was inhibited by dexamethasone and mitogen-activated protein kinase inhibitors. OSM was also found to be biologically active in inducing lung fibroblast proliferation that was partially but significantly inhibited by anti-OSM mAb. In vivo mast cells were found to express OSM in both biopsy specimens and bronchoalveolar lavage fluid from patients with sarcoidosis. CONCLUSION: The production of OSM by human mast cells might represent one link between T cell-induced mast cell activation and the development of a spectrum of structural changes in T cell-mediated inflammatory processes in which mast cells have been found to be involved.

Infevers: an evolving mutation database for auto-inflammatory syndromes.

The Infevers database (http://fmf.igh.cnrs.fr/infevers/) was established in 2002 to provide investigators with access to a central source of information about all sequence variants associated with periodic fevers: Familial Mediterranean fever (FMF), TNF Receptor Associated Periodic Syndrome (TRAPS), Hyper IgD Syndrome (HIDS), Familial Cold Autoinflammatory Syndrome/Muckle-Wells Syndrome/Chronic Infantile Neurological Cutaneous and Articular Syndrome (FCAS/MWS/CINCA). The prototype of this group of disorders is FMF, a recessive disease characterized by recurrent bouts of unexplained inflammation. FMF is the pivotal member of an expanding family of autoinflammatory disorders, a new term coined to describe illnesses resulting from a defect of the innate immune response. Therefore, we decided to extend the Infevers database to genes connected with autoinflammatory diseases. We present here the biological content of the Infevers database, including the introduction of two new entries: Crohn/Blau and Pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA syndrome). Infevers has a range of query capabilities, allowing for simple or complex interrogation of the database. Currently, the database contains 291 sequence variants in related genes (MEFV, TNFRSF1A, MVK, CARD15, PSTPIP1, and CIAS1), consisting of published data and personal communications, which has revealed or refined the preferential mutational sites for each gene. This database will continue to evolve in its content and to improve in its presentation.

Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family.

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) interacts with the EGF-like repeats of the Notch proteins and the EGF precursor.

Employing the yeast two-hybrid system, the Tat protein of the human immunodeficiency virus (HIV) was shown to interact with a region spanning the EGF-like repeats 1-6 of the mouse Notch1, the human Notch2 and the Drosophila Notch. This observation was confirmed in mammalian cells by demonstrating an interaction between the HIV Tat and the EGF-like repeats 1-6 of the various Notch proteins. The HIV Tat protein interacted also with the full-length mouse Notch1 receptor when co-expressed in mammalian cells. Moreover, the HIV Tat protein interacted also with the EGF-like repeats 1-4-spanning domain of the human EGF precursor. The ability of the HIV Tat protein to interact with the Notch proteins and possibly with other EGF-like repeats-bearing proteins, suggests that such interactions might modulate their physiological functions, thus affecting various AIDS-associated pathologies.

Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway.

Pyrin, the familial Mediterranean fever protein, is found in association with the cytoskeleton in myeloid/monocytic cells and modulates IL-1beta processing, NF-kappaB activation, and apoptosis. These effects are mediated in part through cognate interactions with the adaptor protein ASC, which shares an N-terminal motif with pyrin. We sought additional upstream regulators of inflammation by using pyrin as the bait in yeast two-hybrid assays. We now show that proline serine threonine phosphatase-interacting protein [PSTPIP1, or CD2-binding protein 1 (CD2BP1)], a tyrosine-phosphorylated protein involved in cytoskeletal organization, also interacts with pyrin. Recently, PSTPIP1/CD2BP1 mutations were shown to cause the syndrome of pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA), a dominantly inherited autoinflammatory disorder mediated predominantly by granulocytes. Endogenous PSTPIP1/CD2BP1 and pyrin are coexpressed in monocytes and granulocytes and can be coimmunoprecipitated from THP-1 cells. The B box segment of pyrin was necessary and the B box/coiled-coil segment sufficient for this interaction, whereas the SH3 and coiled-coil domains of PSTPIP1/CD2BP1 were both necessary, but neither was sufficient, for pyrin binding. The Y344F PSTPIP1/CD2BP1 mutation, which blocks tyrosine phosphorylation, was associated with a marked reduction in pyrin binding in pervanadate-treated cells. PAPA-associated A230T and E250Q PSTPIP1/CD2BP1 mutations markedly increased pyrin binding as assayed by immunoprecipitation and, relative to WT, these mutants were hyperphosphorylated when coexpressed with c-Abl kinase. Consistent with the hypothesis that these mutations exert a dominant-negative effect on the previously reported activity of pyrin, we found increased IL-1beta production by peripheral blood leukocytes from a clinically active PAPA patient with the A230T PSTPIP1/CD2BP1 mutation and in cell lines transfected with both PAPA-associated mutants.

The Tat protein of the caprine arthritis encephalitis virus interacts with the Notch2 EGF-like repeats and the epithelin/granulin precursor.

Using the yeast two-hybrid system, we screened a human placenta cDNA library and identified two proteins that interacted with the Tat protein of the caprine arthritis encephalitis virus (CAEV): the EGF-like repeats 1-6 of the extracellular domain of the human Notch2 receptor and the epithelin/granulin growth factor precursor. This interaction was also confirmed in mammalian cells. Using in vitro mutagenesis assays, we showed that each one of the three cysteine residues located within the cysteine-rich domain of the CAEV Tat protein is essential for the binding of Tat to both the Notch2 and the epithelin/granulin protein. It is thus suggested that the cysteine-rich domain of Tat plays a role in the interaction between the Tat and either Notch2 or the epithelin/granulin domains, both of which exhibit EGF-like-repeat-imposed spatial conformation. It is assumed that such interactions might modulate the physiological functions of Notch2 and epithelin/granulin, thereby affecting various pathologies associated with CAEV.

The expanding spectrum of systemic autoinflammatory disorders and their rheumatic manifestations.

The authors review the genes, and their respective proteins, responsible for eight autoinflammatory conditions. Familial Mediterranean fever is caused by mutations in pyrin, which is the prototype of a new family of proteins belonging to the death-domain superfamily. This new group of proteins, which regulate apoptosis, inflammation, and cytokine processing, share an approximately 90-amino-acid N-terminal sequence called the PYRIN domain. Mutations in another PYRIN domain protein, termed cryopyrin, are responsible for three clinically defined illnesses, Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and NOMID/CINCA. A related protein encoded by the gene is responsible for the Mendelian disorder, Blau syndrome, and also predisposes to Crohn disease. The gene responsible for PAPA syndrome has recently been identified as, and preliminary results from the authors' laboratory also implicate its protein product in these pathways. Lastly, the authors discuss the broadening genetic and clinical spectrum of TRAPS, an autoinflammatory syndrome resulting from mutations in the 55-kDa receptor for tumor necrosis factor.

De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases.

OBJECTIVE: Neonatal-onset multisystem inflammatory disease (NOMID; also known as chronic infantile neurologic, cutaneous, articular [CINCA] syndrome) is characterized by fever, chronic meningitis, uveitis, sensorineural hearing loss, urticarial skin rash, and a characteristic deforming arthropathy. We investigated whether patients with this disorder have mutations in CIAS1, the gene which causes Muckle-Wells syndrome and familial cold autoinflammatory syndrome, two dominantly inherited disorders with some similarities to NOMID/CINCA syndrome. METHODS: Genomic DNA from 13 patients with classic manifestations of NOMID/CINCA syndrome and their available parents was screened for CIAS1 mutations by automated DNA sequencing. Cytokine messenger RNA (mRNA) levels were assessed by real-time polymerase chain reaction on peripheral blood leukocyte mRNA, and serum cytokine levels were assayed by enzyme-linked immunosorbent assay. Protein expression was assessed by Western blotting of lysates from plastic-adherent peripheral blood mononuclear cells. RESULTS: In 6 of the 13 patients, we found 6 heterozygous missense substitutions in CIAS1. Five of the 6 mutations are novel. None of these sequence changes was observed in a panel of >900 chromosomes from healthy controls. Two distinct nucleotide changes in a single codon in unrelated patients resulted in the same amino acid change. In 4 mutation-positive children whose parental DNA was available, no mutation was found in the parental DNA, supporting the conclusion that the mutations arose de novo. Consistent with the recently discovered role of CIAS1 in the regulation of interleukin-1 (IL-1), we found evidence of increased IL-1beta, as well as tumor necrosis factor, IL-3, IL-5, and IL-6, but not transforming growth factor beta, in a mutation-positive patient compared with normal controls. CONCLUSION: Our data increase the total number of known germline mutations in CIAS1 to 20, causing a spectrum of diseases ranging from familial cold autoinflammatory syndrome to Muckle-Wells syndrome to NOMID/CINCA syndrome. Mutations in CIAS1 were only found in approximately 50% of the cases identified clinically as NOMID/CINCA syndrome, which raises the possibility of genetic heterogeneity. IL-1 regulation by CIAS1 suggests that IL-1 receptor blockade may constitute a rational approach to the treatment of NOMID/CINCA syndrome.