Alternative titles; symbols
HGNC Approved Gene Symbol: CARD8
Cytogenetic location: 19q13.33 Genomic coordinates (GRCh38): 19:48,179,825-48,255,946 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 19q13.33 | ?Inflammatory bowel disease (Crohn disease) 30 | 619079 | Autosomal dominant | 3 |
Caspase recruitment domain (CARD)-containing proteins, such as CARD8, are involved in pathways leading to activation of caspases or nuclear factor kappa-B (NFKB; see 164011) in the context of apoptosis or inflammation, respectively (Bouchier-Hayes et al., 2001).
By sequencing clones obtained from a size-fractionated human brain cDNA library, Nagase et al. (1999) cloned CARD8, which they designated KIAA0955. The transcript contains several repetitive elements in the 3-prime UTR, and the deduced protein contains 431 amino acids. RT-PCR ELISA detected highest expression of CARD8 in kidney and corpus callosum and lowest expression in pancreas. All other tissues and specific brain regions examined showed intermediate expression.
By searching databases for sequences similar to the CARD domain of APAF1 (602233), followed by PCR, Pathan et al. (2001) cloned CARD8, which they designated TUCAN. CARD8 contains an N-terminal segment that shares 50% amino acid identity with a region of the proapoptotic protein DEFCAP (NALP1; 606636), as well as a C-terminal CARD domain.
By Western blot analysis of several tissues, Bouchier-Hayes et al. (2001) found CARD8 expressed at an apparent molecular mass of 50 kD, close to the predicted molecular mass of 49 kD. Highest expression was in lung, ovary, testis, and placenta, with low or absent expression in brain, skeletal muscle, and spleen.
By RT-PCR, Razmara et al. (2002) found highest CARD8 expression in placenta, spleen, lymph node, and bone marrow.
By EST analysis, Zhang and Fu (2002) identified 5 splice variants of CARD8, which they called NDPP1. The variants all have different translation start sites.
Yamamoto et al. (2005) cloned a splice variant of CARD8 that they named TUCAN54 after the calculated molecular mass of the encoded protein. The deduced 487-amino acid TUCAN54 protein has a unique 80-amino acid N terminus compared with the 48-kD isoform, TUCAN48, but both proteins contain a NALP homology domain, a candidate caspase cleavage site (DEED), a C-terminal CARD domain, and several putative phosphorylation sites. The N terminus of TUCAN54 provides phosphorylation sites not found in TUCAN48. RT-PCR detected high TUCAN54 expression in leukocytes and spleen. Expression was lower in heart, lung, thymus, liver, pancreas, and testis, and little to no expression was detected in other tissues examined. TUCAN54 was widely expressed in a variety of tumor cell lines.
By EST database and RT-PCR analyses, Bagnall et al. (2008) characterized 5 isoforms of CARD8 that differ in their N termini and have predicted molecular masses of 47.5, 48, 51, 54, and 60 kD. The major 48-kD isoform has 432 amino acids and starts in exon 5, and the 54-kD isoform has 487 amino acids and starts in exon 4. The 47.5-kD isoform differs in the first 20 amino acids from the 48-kD isoform and results from a putative initiation codon 20 bp upstream of exon 6. Western blot analysis of lymphoblastoid cell lines from 6 Crohn disease (see IBD1; 266600) patients showed a 48- or 47.5-kD band in all cell lines and an additional band of 54 kD in only 1 cell line.
Zhang and Fu (2002) determined that the CARD8 gene contains 14 exons and spans more than 50 kb. The first 3 exons are noncoding.
By genomic sequence analysis, Zhang and Fu (2002) mapped the CARD8 gene to chromosome 19q13.3.
By overexpression in Jurkat human T cells, Pathan et al. (2001) found that CARD8 inhibited apoptosis and caspase activation induced by APAF1- and CASP9 (602234)-dependent stimuli but not APAF1- and CASP9-independent stimuli. Immunohistochemical analysis detected elevated CARD8 immunostaining in 42 of 66 (64%) colon cancer specimens compared with the adjacent normal tissues. Higher endogenous CARD8 immunostaining correlated with shorter patient survival.
Bouchier-Hayes et al. (2001) found that CARD8 suppressed NFKB activation associated with overexpression of NFKB activators or with ligand-induced stimulation of IL1 receptor (see 147810) or TNF receptor (see 191190). Coimmunoprecipitation experiments revealed that CARD8 interacted with the regulatory subunit of the I-kappa-B kinase complex, IKK-gamma (IKBKG; 300248). Bouchier-Hayes et al. (2001) concluded that CARD8 is a regulator of NFKB activation in the context of proinflammatory signals.
Razmara et al. (2002) found that CARD8 mediated apoptosis. Overexpression of CARD8 induced apoptosis in transfected breast cancer and green monkey kidney cells. In contrast to the findings of Pathan et al. (2001), inhibitor studies by Razmara et al. (2002) indicated that apoptosis was induced by CARD8 via the APAF1/CASP9 apoptotic complex. CARD8 also suppressed NFKB activation by diverse stimuli, and stable CARD8 expression sensitized monocytes to differentiation-induced apoptosis. Razmara et al. (2002) found that CARD8 bound CASP1 (147678) and negatively regulated CASP1-dependent IL1B (147720) secretion in monocytes. In addition, CARD8 bound the CASP1 inhibitors ICEBERG (605354) and pseudo-ICE. Razmara et al. (2002) concluded that CARD8 may be an adaptor molecule that regulates CASP1 activation, NFKB activation, and apoptosis.
Zhang and Fu (2002) found that expression of CARD8 blocked BAX (600040)-induced apoptosis in several human cell lines and in rat embryonic fibroblasts. TNF-alpha-induced NFKB activation was suppressed in a CARD8-transfected hepatoma cell line.
Agostini et al. (2004) noted that NALP1, unlike other short NALP proteins, contains a C-terminal CARD domain that interacts with and activates CASP5 (602665). CASP1 and CASP5 are activated when they assemble with NALP1 and ASC (PYCARD; 606838) to form the inflammasome, which is responsible for processing the inactive IL1B precursor (proIL1B) to release active IL1B cytokine. Using immunoprecipitation analysis, Agostini et al. (2004) found that CARD8, which contains C-terminal FIIND (function to find) and CARD domains, associated with constructs of NALP2 (609364) and NALP3 (NLRP3; 606416) lacking the N-terminal pyrin domain and/or the C-terminal leucine-rich repeat domain. They determined that the interaction was mediated by the FIIND domain of CARD8 and the centrally located NACHT domain of NALP2 and NALP3. The pyrin domain of NALP2 and NALP3, like that of NALP1, interacted with the pyrin domain of ASC, which recruits CASP1. Transfection experiments showed that an inflammasome could be assembled containing ASC, CARD8, CASP1, and a short NALP, resulting in activation of CASP1, but not CASP5, and strong processing of proIL1B.
Yamamoto et al. (2005) found that overexpression of TUCAN54 in human cell lines inhibited pro-CASP9 activation and suppressed apoptosis induced by staurosporin, a protein kinase inhibitor, and by etoposide, a chemotherapeutic reagent. In contrast, suppression of TUCAN54 expression via small interfering RNA increased etoposide-induced cell death. TUCAN54 also inhibited CASP8 (601763) activation, thereby suppressing FAS (TNFRSF6; 134637)-induced cell death. TUCAN48 inhibited CASP9 activation and, more weakly, CASP8 activation, but only TUCAN54 physically associated with FADD (602457). FADD constitutively associated with pro-CASP8 in transfected cells, suggesting that TUCAN54 inhibits pro-CASP8 activation by forming a molecular complex with FADD and pro-CASP8.
In HEK293 cells transfected with plasmids expressing either CARD8 T60 or T48 isoforms, Mao et al. (2018) observed significantly reduced levels of IL1B compared to cells transfected with empty vector. The authors concluded that both isoforms of CARD8 play an inhibitory role during NLRP3 inflammasome activation.
Sharif et al. (2021) determined the cryoelectron microscopy structure of the CARD8 CARD8-DPP9 (608258) complex at 3.3-angstrom resolution. The structure contained 2 copies of CARD8 bound to a single DPP9 subunit, forming a CARD8A-CARD8B-DPP9 ternary complex in which CARD8A was full-length CARD8, and CARD8B was the autoproteolyzed CARD8 C-terminal fragment (CT) consisting of the UPA subdomain of the FIIND domain and the CARD domain of CARD8. Unlike the NLRP1 (606636)-DPP9 complex, the final CARD8-DPP9 complex had a 2:2 CARD8:DPP9 stoichiometry in which 1 monomer of the DPP9 dimer was not bound to CARD8. Moreover, the N terminus of the UPA subdomain of the CARD8B molecule did not insert into the DPP9 active site and was largely disordered. Similar to the NLRP1-DPP9 complex, assembly of the CARD8-DPP9 complex was mediated by interfaces between ZU5 of CARD8A and DPP9, between UPA of CARD8B and DPP9, and between the UPA subdomains of CARD8A and CARD8B. Mutation analysis revealed that the interface between UPA of CARD8B and DPP9 was crucial for ternary complex formation, whereas the interface between the UPA subdomains of CARD8A and CARD8B was critical for CARD8 inflammasome signaling. Unlike NLRP1-DPP9 assembly, in which both copies of NLRP1 are required for DPP9 association, binding of CARD8A to DPP9 did not require CARD8B. Nevertheless, DPP9 binding restrained CARD8B-CT in the complex following recruitment of CARD8B-CT by full-length CARD8A, thereby preventing inflammasome formation. Since the N terminus of CARD8-CT did not bind the DPP9 active site, binding of DPP9 to CARD8-CT was not directly affected by the DPP9 inhibitor Val-boroPro. However, Val-boroPro still activated the CARD8 inflammasome by promoting CARD8 N-terminal degradation and by weakening ternary complex stability.
In a boy, his mother, and maternal aunt with Crohn disease (IBD30; 619079), Mao et al. (2018) identified heterozygosity for a missense mutation in the CARD8 gene (V44I; 609051.0001). Functional analysis showed that the mutant protein exerts a dominant-negative effect by forming oligomers with unmutated T60 and T48 CARD8 isomers, which impedes binding to NLRP3 (606416). The authors did not detect a reported Crohn disease-associated CARD8 SNP (rs2043211) in the proband or his parents.
Associations Pending Confirmation
Using RNA from lymphoblastoid cell lines of Crohn disease patients, Bagnall et al. (2008) showed that an A-to-T transversion in exon 5 of the CARD8 gene (rs2043211), predicted to result in a cys10-to-ter (C10X) substitution in the 48-kD isoform of CARD8, did not affect the 47.5-kD isoform. Patients who were TT homozygous showed somewhat reduced expression of CARD8 mRNA, but expressed a 47.5-kD protein. The authors showed that the rs2043211 variant had multiple outcomes including unaffected, cys10 to ter, cys34 to ter, phe52 to ile, and phe102 to ile. Bagnall et al. (2008) noted that the multiple isoforms and differing consequences for a predicted stop codon polymorphism underline the importance of detailed analysis of the effects of proposed functional variants on gene expression.
Using a genomewide screen of Salmonella typhimurium-infected HapMap lymphoblastoid cells from individuals of northern and western European or Nigerian ancestry, Ko et al. (2009) identified a loss-of-function allele of CARD8, C10X, that was associated with increased cell death in vitro. Overexpression of alternative alleles and RNA interference analysis supported the association. Genotyping of individuals with systemic inflammatory response syndrome (SIRS) showed a modestly increased risk with the variant. Ko et al. (2009) proposed that loss of CARD8 function and an increased inflammatory response may protect against Salmonella but lead to increased inflammatory disease.
Eklund et al. (2014) used Mycobacterium tuberculosis (see 607948) to infect macrophages of individuals who had inflammatory disease and associated polymorphisms in NLRP3 (met299 to val (M299V) or gln705 to lys (Q705K)) or in both NLRP3 and CARD8 (C10X). In individuals with combined NLRP3 and CARD8 variants, the authors observed restricted bacterial growth in cells. The variants, in combination, led to constitutive secretion of IL1B, elevated IL1B levels after infection, and enhanced CD63 (155740)-positive phagolysosomal fusion. Restricted growth was also observed in healthy blood donors who had variants in both genes (Q705K in NLRP3 and C10X in CARD8), but not in those carrying only 1 of the variants. Eklund et al. (2014) concluded that gain-of-function variants in NLRP3 (i.e., M299V or Q705K) in combination with the C10X variant in CARD8 result in superior control of M. tuberculosis growth.
In a boy, his mother, and his maternal aunt with Crohn disease (IBD30; 619079), Mao et al. (2018) identified heterozygosity for a C-to-T transition in exon 5 of the CARD8 gene (chr19.48,741,719C-T, GRCh37), resulting in a val44-to-ile (V44I) substitution in the T60 isoform. The mutation, which was not found in the boy's unaffected father, was present in the gnomAD database at a minor allele frequency of 0.0015%. Proband serum showed increased levels of the cytokines IL1B (147720) and IL6 (147620) compared to sex-matched control sera. In addition, supernatants from proband peripheral blood mononuclear cells and monocyte cultures had significantly increased amounts of IL1B compared to controls, consistent with dysregulation of the NLRP3 (606416) inflammasome. Cells from the proband's affected mother and aunt displayed similar overactivity, suggesting that the V44I mutation negates the inhibitory role of CARD8 on NLRP3 inflammasome activation. Experiments with transfected HEK293 cells demonstrated that the V44I mutant exerts a dominant-negative effect by binding to and forming oligomers with unmutated T60 or T48 CARD8, which impedes binding to NLRP3. Additional studies suggested that overactivation of the NLRP3 inflammasome in CD patients with the V44I mutant is caused by both reduced phosphorylation and reduced polyubiquitination of NLRP3.
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Bagnall, R. D., Roberts, R. G., Mirza, M. M., Torigoe, T., Prescott, N. J., Mathew, C. G. Novel isoforms of the CARD8 (TUCAN) gene evade a nonsense mutation. Europ. J. Hum. Genet. 16: 619-625, 2008. [PubMed: 18212821] [Full Text: https://doi.org/10.1038/sj.ejhg.5201996]
Bouchier-Hayes, L., Conroy, H., Egan, H., Adrain, C., Creagh, E. M., MacFarlane, M., Martin, S. J. CARDINAL, a novel caspase recruitment domain protein, is an inhibitor of multiple NF-kappa-B activation pathways. J. Biol. Chem. 276: 44069-44077, 2001. [PubMed: 11551959] [Full Text: https://doi.org/10.1074/jbc.M107373200]
Eklund, D., Welin, A., Andersson, H., Verma, D., Soderkvist, P., Stendahl, O., Sarndahl, E., Lerm, M. Human gene variants linked to enhanced NLRP3 activity limit intramacrophage growth of Mycobacterium tuberculosis. J. Infect. Dis. 209: 749-753, 2014. [PubMed: 24158955] [Full Text: https://doi.org/10.1093/infdis/jit572]
Ko, D. C., Shukla, K. P., Fong, C., Wasnick, M., Brittnacher, M. J., Wurfel, M. M., Holden, T. D., O'Keefe, G. E., Van Yserloo, B., Akey, J. M., Miller, S. I. A genome-wide in vitro bacterial-infection screen reveals human variation in the host response associated with inflammatory disease. Am. J. Hum. Genet. 85: 214-227, 2009. [PubMed: 19664744] [Full Text: https://doi.org/10.1016/j.ajhg.2009.07.012]
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Razmara, M., Srinivasula, S. M., Wang, L., Poyet, J.-L., Geddes, B. J., DiStefano, P. S., Bertin, J., Alnemri, E. S. CARD-8 protein, a new CARD family member that regulates caspase-1 activation and apoptosis. J. Biol. Chem. 277: 13952-13958, 2002. [PubMed: 11821383] [Full Text: https://doi.org/10.1074/jbc.M107811200]
Sharif, H., Hollingsworth, L. R., Griswold, A. R., Hsiao, J. C., Wang, Q., Bachovchin, D. A., Wu, H. Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment. Immunity 54: 1392-1404, 2021. [PubMed: 34019797] [Full Text: https://doi.org/10.1016/j.immuni.2021.04.024]
Yamamoto, M., Torigoe, T., Kamiguchi, K., Hirohashi, Y., Nakanishi, K., Nabeta, C., Asanuma, H., Tsuruma, T., Sato, T., Hata, F., Ohmura, T., Yamaguchi, K., Kurotaki, T., Hirata, K., Sato, N. A novel isoform of TUCAN is overexpressed in human cancer tissues and suppresses both caspase-8- and caspase-9-mediated apoptosis. Cancer Res. 65: 8706-8714, 2005. [PubMed: 16204039] [Full Text: https://doi.org/10.1158/0008-5472.CAN-04-4649]
Zhang, H., Fu, W. NDPP1 is a novel CARD domain containing protein which can inhibit apoptosis and suppress NF-kappa-B activation. Int. J. Oncol. 20: 1035-1040, 2002. [PubMed: 11956601]