Functional Studies of Cryopyrin V200M Mutation Identified in Beh

1
Functional Studies of Cryopyrin V200M Mutation
Identified in Behçets Disease
Patients
PP-078-10
Elif Eren, Yetis Gültekin, Sahru Yüksel and
Nesrin Özören
Bogaziçi University, Department of Molecular
Biology and Genetics, Kuzey Park Binasi, 34342
Bebek Istanbul-TURKEY Phone  [phone]
29  Fax  [phone], e-mails
eliferen2_at_gmail.com, yetisgultekin_at_yahoo.com

• 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-IL1ß
  into mature IL-1ß that can be secreted and can
  initiate innate responses against the pathogen.
  Active inflammasomes can also activate the NF-?B
  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-?B pathway with
  luciferase reporter gene assays.

FIGURE 2 Genetic Studies
FIGURE 1 Inflammasome and Behçets Disease

• Inflammasome cryopyrin ASC pro-caspase-1
• Pathogens infection pro-caspase-1 ? caspase-1
  ? IL1ß secretion ? inflammation

• Screening of Behçets patients cryopyrin and ASC
  genes for mutations.

• 1344 G?A G/A heterozygote
• 200 GTG ? ATG
• Valine
  Methionine

• Identification of cryopyrin V200M mutation in 3
  patients among 103.
• Not found in 50 controls.

• Nucleotide conserved among species
• ? important role for protein function

• Pedigree analysis

From Mariathasan S., ASC, Ipaf and
Cryopyrin/Nalp3 bona fide intracellular adapters
of caspase-1 inflammasome,
Microbes and Infection 9
(2007) 664-671.
No mutation found in healthy family members.

• Behçets disease auto-inflammatory disease
• Activation of inflammasome even in absence of
  pathogens.
• Hypothesis
• Mutation in inflammasome component
• ? over-activation of inflammasome

• Polymorphism analysis of cryopyrin exon 3

FIGURE 3 Transfection of HEK293FT cells with
different plasmids
Highly polymorphic exon
HEK293FT cells were transfected via Ca3(PO4)2
with 70 efficiency
FIGURE 5 Effect of V200M Mutation on
Inflammasome Assembly and Function
FIGURE 4 The effect of V200M mutation on NF-kB
activity- Preliminary Results
Interaction with ASC
Caspase-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-immunoprecipitati
on is performed.
Caspase-1 activation is compared by Western blot
between HEK293T cells transfected with Caspase-1,
ASC and WT or mutant Cryopyrin.
Cryo WT 10 ng
Cryo V200M 10 ng 10 ng
ASC 200 ng 200 ng
Nod 1 100 ng

• 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.

ACKNOWLEDGMENTS This project was supported by
TÜBA-GEDIP award, EMBO-YIP-SDIG 1468 to N.Ö and
BAP-06HB103 to S.Y. Y.G is supported by Turkish
Education Foundation Scholarship (T.E.V) Congress
attendance was partly funded by Bogaziçi
University Arts and Science Faculty and The
Institute of Science.