Presentation on theme: "PP-078-10 Functional Studies of Cryopyrin V200M Mutation Identified in Behçet’s Disease Patients Elif Eren, Yetiş Gültekin, Şahru Yüksel."— Presentation transcript:
1PPFunctional Studies of Cryopyrin V200M Mutation Identified in Behçet’s Disease PatientsElif Eren, Yetiş Gültekin, Şahru Yüksel and Nesrin ÖzörenBoğaziçi University, Department of Molecular Biology and Genetics, Kuzey Park Binası,34342 Bebek Istanbul-TURKEYPhone: + Fax: + , s:ABSTRACTThe inflammasome is a cytoplasmic complex composed of ASC, NALP3 (cryopryrin) and caspase-1, which are assembled in response to pathogens. This assembly leads to caspase-1 cleavage. Active caspase-1 then cleaves pro-IL1β into mature IL-1β that can be secreted and can initiate innate responses against the pathogen. Active inflammasomes can also activate the NF-κB pathway. It has been previously shown that mutations in components of this complex cause auto-inflammatory diseases such as familial cold autoinflammatory syndrome (FACS), Muckle-Wells syndrome (MWS), and neonatal-onset multiple-system inflammatory disease (NOMID). Behçet’s disease is also an auto-inflammatory disease of unknown etiology characterized by recurrent inflammatory episodes. Aberrant secretion of cytokines is observed in Behçet’s disease. Thus, we hypothesized that mutations in inflammasome components may result in a constitutively active complex. We have identified cryopyrin V200M mutation in a higher frequency (3/103 patients, not found in 50 controls) than previously reported. To test if cryopyrin V200M mutation results in a constitutively active inflammasome, we have cloned the mutation into vector and we are investigating whether this mutation over-activates NF-κB pathway with luciferase reporter gene assays.FIGURE 1: Inflammasome and Behçet’s DiseaseFIGURE 2: Genetic StudiesInflammasome: cryopyrin + ASC + pro-caspase-1Pathogens infection: pro-caspase-1 caspase IL1β secretion inflammationScreening of Behçet’s patients cryopyrin and ASC genes for mutations.1344 GA G/A heterozygote200 GTG ATGValine MethionineIdentification of cryopyrin V200M mutation in 3 patients among 103.Not found in 50 controls.Nucleotide conserved among species important role for protein functionPedigree analysis:From Mariathasan S., ASC, Ipaf and Cryopyrin/Nalp3: bona fide intracellular adapters of caspase-1 inflammasome, Microbes and Infection 9 (2007)No mutation found in healthy family members.Behçet’s disease: auto-inflammatory diseaseActivation of inflammasome even in absence of pathogens.Hypothesis:Mutation in inflammasome component over-activation of inflammasomePolymorphism analysis of cryopyrin exon 3FIGURE 3: Transfection of HEK293FT cells with different plasmidsHighly polymorphic exonHEK293FT cells were transfected via Ca3(PO4)2 with 70% efficiencyFIGURE 4: The effect of V200M mutation on NF-kB activity- Preliminary ResultsFIGURE 5: Effect of V200M Mutation on Inflammasome Assembly and FunctionInteraction with ASCCaspase-1 ActivationIn order to determine if V200M mutation affects interaction between Cryopyrin and ASC, HEK293T cells are transfected with different plasmids expressing these proteins and co-immunoprecipitation is performed.Caspase-1 activation is compared by Western blot between HEK293T cells transfected with Caspase-1, ASC and WT or mutant Cryopyrin.Cryo WT10 ngCryo V200MASC200 ngNod 1100 ngCONCLUSIONSCryopyrin V200M mutation was found in 3/103 Behcet’s disease patients.Many polymorphisms were identified in Cryopyrin exon 3 in the general Turkish population.Preliminary results show that NF-kB activity increases 2 fold with V200M Cryopyrin compared to WT.Experiments are ongoing to test if mutant protein’s interaction with its partners ASC and caspase-1 increases by co-ip.ACKNOWLEDGMENTSThis project was supported by TÜBA-GEDIP award, EMBO-YIP-SDIG 1468 to N.Ö and BAP-06HB103 to Ş.Y.Y.G is supported by Turkish Education Foundation Scholarship (T.E.V)Congress attendance was partly funded by Boğaziçi University Arts and Science Faculty and The Institute of Science.