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AN EVOLUTIONARILY CONSERVED ROLE FOR ANKYRIN DOMAINS IN INFLAMMASOME ACTIVATION
Autores:
SYLWIA DOMINIKA TYRKALSKA
, SERGIO CANDEL CAMACHO,
ANA VALERA PÉREZ
, ANA PEREZ OLIVA,
FRANCISCA ALCARAZ PÉREZ
,
DIANA GARCIA MORENO
,
MARIA LUISA CAYUELA FUENTES
,
VICTORIANO MULERO MÉNDEZ
,
Grupos de investigación:
[GI/IMIB/C060/2011] Inmunidad, inflamación y cáncer
[GI/IMIB/C003/2011] Cirugía digestiva, endocrina y trasplante de órganos abdominales
Comunicación:
Antecedentes:
Many proteins contain tandemly repeated modules of several amino acids, which act as the building blocks that form the underlying architecture of a specific protein-binding interface. Among these motifs and one of the most frequently observed are ankyrin repeats (ANK), which consist of 33 amino acid residues that are highly conserved. ANK domains span a wide range of functions, including protein-protein interactions, such as the recruitment of substrate to the catalytic domain of an enzyme, or the assembly of stable multiprotein complexes.
Métodos:
Microinjection: Morpholinos (0.5–1.0 ng/egg) and mRNAs (200 ng/µl of injection mix) were microinjected (1 nl) into the yolk sac of one-cell-stage embryos using a Narishige IM300 microinjector. Caspase-1 activity: 25-30 larvae were collected and homogenized at 72 hours post-fertilization (hpf). Cells were lysed and the protein concentration was measured using a BCA commercial kit. The same amount of total proteins for each sample was used to measure caspase-1 activity with the fluorometric substrate Z-YVAD-AFC by FLUOstart spectrofluorometer (BGM). Infection - Survival: In all cases, 100-150 larvae were infected with Salmonella Typhimurium by the injection of a MOI of 10 in the yolk sac at 48 hpf. Those larvae had been previously injected with the morpholinos/mRNAs. Survival curves were made after counting the number of surviving larvae during 5 days after the infection. RT-qPCR: Total RNA was extracted from 20-25 pooled larvae at 72 hpf with TRIzol® Plus RNA Purification System (Invitrogen). Real-time PCR was performed with an ABI PRISM 7500 instru- ment (Applied Biosystems) using SYBR Green PCR Core Reagents (Applied Biosystems). Cell-Sorting:Approximately 300 to 500 non-infected and infected larvae from the lines Tg(mpx:eGFP) and Tg(mpeg1:eGFP) were anesthetized in tricaine at 24 hpi, minced with a razor blade, incubated at 28°C for 30 min with 0.077 mg/ml Liberase (Roche) and the resulting cell suspension passed through a 40 µm cell strainer. Cell sorting was performed on a FACSCalibur (BD Biosciences) and a SH800Z (Sony). WISH: Transparent Casper embryos were used for WISH (51). caiap RNA probes were generated using the DIG RNA Labelling Kit (Roche Applied Science) from linearized plasmids. Embryos were imaged using a Scope.A1 stereomicroscope equipped with a digital camera (AxioCam ICc 3, Zeiss).
Resultados:
Here we report the identification of an evolutionarily conserved protein, that we term Caiap (from CARD- and ANK-containing Inflammasome Adaptor Protein), which has an N-terminal CARD domain and 16 C-terminal ANK domains and is required for the inflammasome-dependent clearance of Salmonella Typhimurium in zebrafish by macrophages. Intriguingly, Caiap is highly conserved from cartilaginous fish to marsupials but is absent in placental mammals. Despite the presence of the CARD domain, Caiap requires the universal inflammasome adaptor Asc to mediate its antibacterial function. Surprisingly, Caiap physically interacts with active Caspa, the functional homologue of mammalian caspase-1, through its ANK domain.
Conclusiones:
Our results therefore point to ANK domain-containing proteins as key inflammasome adaptors that are required for its effector functions.
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