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FIGURE 1: Inflammasome and Behçets Disease Functional Studies of Cryopyrin V200M Mutation Identified in Behçets Disease Patients Elif Eren, Yetiş Gültekin,

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Presentation on theme: "FIGURE 1: Inflammasome and Behçets Disease Functional Studies of Cryopyrin V200M Mutation Identified in Behçets Disease Patients Elif Eren, Yetiş Gültekin,"— Presentation transcript:

1 FIGURE 1: Inflammasome and Behçets Disease Functional Studies of Cryopyrin V200M Mutation Identified in Behçets Disease Patients Elif Eren, Yetiş Gültekin, Şahru Yüksel and Nesrin Özören Boğaziçi University, Department of Molecular Biology and Genetics, Kuzey Park Binası, Bebek Istanbul-TURKEY Phone: Fax: , s: ABSTRACT The 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). Behçets disease is also an auto-inflammatory disease of unknown etiology characterized by recurrent inflammatory episodes. Aberrant secretion of cytokines is observed in Behçets 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 2: Genetic Studies CONCLUSIONS Cryopyrin V200M mutation was found in 3/103 Behcets 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 proteins interaction with its partners ASC and caspase-1 increases by co-ip. Inflammasome: cryopyrin + ASC + pro-caspase-1 Pathogens infection: pro-caspase-1 caspase-1 IL1β secretion inflammation Behçets disease: auto-inflammatory disease Activation of inflammasome even in absence of pathogens. Hypothesis: Mutation in inflammasome component over-activation of inflammasome Screening of Behçets patients cryopyrin and ASC genes for mutations. Identification of cryopyrin V200M mutation in 3 patients among 103. Not found in 50 controls. Nucleotide conserved among species important role for protein function 1344 G A G/A heterozygote 200 GTG ATG Valine Methionine Pedigree analysis: FIGURE 3: Transfection of HEK293FT cells with different plasmids ACKNOWLEDGMENTS This 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. FIGURE 5: Effect of V200M Mutation on Inflammasome Assembly and Function PP Polymorphism analysis of cryopyrin exon 3 Highly polymorphic exon No mutation found in healthy family members. From Mariathasan S., ASC, Ipaf and Cryopyrin/Nalp3: bona fide intracellular adapters of caspase-1 inflammasome, Microbes and Infection 9 (2007) FIGURE 4: The effect of V200M mutation on NF-kB activity- Preliminary Results HEK293FT cells were transfected via Ca 3 (PO 4 ) 2 with 70% efficiency Interaction with ASCCaspase-1 Activation In 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 ng Cryo V200M10 ng ASC200 ng Nod 1100 ng


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