CARD19, the protein formerly known as BinCARD, is a mitochondrial protein that does not regulate Bcl10-dependent NF-κB activation after TCR engagement

Abstract

After T cell receptor (TCR) engagement, the CARD11-Bcl10-Malt1 (CBM) complex oligomerizes to transduce NF-κB activating signals. Bcl10 is then degraded to limit NF-κB activation. The cDNA AK057716 (BinCARD-1) was reported to encode a novel CARD protein that interacts with Bcl10 and modestly inhibits NF-κB activation. In a later study, a second isoform, BinCARD-2, was identified. Here, we report that the cDNA AK057716 (BinCARD-1) is an incompletely spliced derivative of the gene product of C9orf89, whereas CARD19 (BinCARD-2) represents the properly spliced isoform, with conservation across diverse species. Immunoblotting revealed expression of CARD19 in T cells, but no evidence of BinCARD-1 expression, and microscopy demonstrated that endogenous CARD19 localizes to mitochondria. Although we confirmed that both BinCARD-1 and CARD19 can inhibit NF-κB activation and promote Bcl10 degradation when transiently overexpressed in HEK293T cells, loss of endogenous CARD19 expression had little effect on Bcl10-dependent NF-κB activation, activation of Malt1 protease function, or Bcl10

Genetic targeting ofCard19is linked to disruptedNinj1 expression, impaired cell lysis, and increased susceptibility toYersiniainfection

“…These data indicate that Card19 -/- cells most likely do not have a defect at the level of GSDMD-dependent pore formation and cytokine release and that the Card19 -/- cells decouples secretion of IL-1β from pyroptosis, which normally occurs in response to these stimuli (26, 32). Consistent with a recent finding that Card19 -/- cells do not have altered NF-κB activation (33), Card19 -/- BMDMs did not have a defect in secretion of IL-6, TNF, or IL-12p40, (Fig. 6C-6E ) .…”

“…Passenger mutations in genes closely linked with the gene of interest in backcrossed C57BL/6 mice can confound interpretation of immune responses and cell death pathways in these lines (23, 84, 85). Notably, Card19 -/- mice were originally generated using 129SvEvBrd ESCs, and backcrossed for 10-12 generations (33). SNP genotyping analysis of the Card19 -/- mice indicated that approximately six megabases adjacent to the Card19 locus were derived from the 129SvEvBrd lineage (Fig.…”

“…Second, BMDMs from numerous independent Card19 +/- BMDMs exhibit wild-type phenotypes, indicating that homozygosity at the Card19 locus is required for the phenotype. Third, SNP analysis reveals only a 6MB region adjacent to Card19 as being derived from the 129SvEvBrd ES cells used to generate the original Card19 -/- line (33). While it is possible that disruption of Card19 in this line also inadvertently impacts NINJ1 expression, our RNA-seq and western blot analyses indicate minimal, if any, impact on Ninj1 mRNA and protein levels in Card19 -/- BMDMs.…”

“…Card19 -/- mice were previously described (33) and maintained as a breeding line in-house. B6 and Casp1 / Casp11 -/- mice obtained from Jackson Laboratories and subsequently maintained as a breeding line in-house.…”

“…Numerous mitochondrial proteins have been implicated in recent years as essential regulators of caspase activity, GSDMD and terminal events in during lysis (35,37,80). While these studies were in progress, Sterile alpha and armadillo motif-containing protein (SARM1) was found to regulate terminal cell lysis following stimulation of the NLRP3 inflammasome (41), and like CARD19, also localizes to the mitochondrial outer membrane (33). Unexpectedly however, BMDMs isolated from multiple independent lines of Sarm1 -/mice (described further in Materials and Methods), including the Sarm1 -/mice reported in the Carty et al study (41), originally described by Kim et al 81, did not exhibit any defects in their ability to undergo cell lysis in response to canonical NLRP3 inflammasome stimuli (Fig.…”