In addition to this nodosome, multiprotein module, another major type of NLR complex, the inflammasome, is formed by pyrin domain-containing NLR proteins, e.g. NALP1 and NALP3 and several adaptor molecules, e.g. ASC and CARD8 (12,�C14). The protein complex selleck chemicals llc forms a large scaffold, which is required for caspase-1-dependent IL-1�� processing and secretion (15, 16). CARD8 (also known as TUCAN and CARDINAL) is comprised of a C-terminal CARD domain and a N-terminal FIIND (domain with function to find) domain (17). CARD8 was first described in the context of NALP-independent NF-��B activation and apoptosis (17, 18). It has been reported to form dimers and bind to procaspase-9 suppressing caspase-9 activation via Apaf1-dependent mechanisms (19). However, CARD8 overexpression has been shown to induce apoptosis (20).

Interestingly CARD8 expression is elevated in colonic carcinoma cells (19) and correlates with shorter patient survival (19, 21) indicating an involvement in cancer progression. CARD8 has been postulated to serve as a molecular bridge recruiting an additional caspase-1 molecule to the NALP3 inflammasome through homotypic CARD-CARD-interaction (13). Thus, upon activation CARD8 may bind with its FIIND domain to the central NBD part of NALP3. Based on structural homology of NALP3 and NOD2, we investigated the possible interaction of CARD8 and NOD2. We hypothesized that the interaction of CARD8 with NOD2 may have an impact on cellular regulation of caspase-1 and NF-��B activation in the context of innate immune reactions.

EXPERIMENTAL PROCEDURES Construction of Plasmids The generation of FLAG-tagged constructs containing full-length wild-type NOD2, the NBD, the LRR, and the CARD domains of NOD2 have been described previously (22). Expression constructs for Myc- and CFP-tagged NOD2, YFP-tagged CARD8, and GFP-tagged CARD8 (1�C320) were generated using standard cloning techniques (see supplemental Table S1). Plasmids pcDNA3.1-CARD8 and pEGFPC3-CARD8 have been described elsewhere (17). The plasmids pFLAG-CMV-caspase1 and pcDNA3-murine-proIL-1�� were a kind gift from Dr. Junying Yuan. RNAi Knockdown of CARD8 Control and specific siRNAs against CARD8 were purchased from Invitrogen (Carlsbad, CA; supplemental Table S3). Transfection of HeLaS3 cells with a pool of three different siRNAs was performed using siPort Amine (Applied Biosystems, Foster City, CA).

Knockdown efficiency was evaluated by semi-quantitative RT-PCR. Immunoprecipitation and Western Blot Transfected HEK293 cells were subjected to co-immunoprecipitation according to standard protocols Carfilzomib (23). Total protein lysates were prepared as described previously (24, 25). Proteins were transferred onto 0.45 ��m polyvinylidene difluoride membranes (Millipore, Billerica, MA) and probed with respective antibodies.