Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses

Abstract

Proteins of the NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] family may serve as critical pathogen-sensing and signal-transducing molecules within the innate immune system. In the present paper, we show that CLAN [CARD (caspase-recruitment domain), LRR (leucine-rich repeat) and NACHT domain-containing protein], a NACHT-containing protein originally demonstrated to bind and activate pro-caspase 1, is also capable of influencing the functions of other members of the NACHT family. Through heterotypic NACHT-domain interactions, CLAN was found to associate with Nod1, Nod2 and NAC [nucleotide-binding domain and CARD-containing protein; NALP1 (NACHT, LRR and PYRIN protein 1)] when co-expressed in HEK-293T (human embryonic kidney) cells. NF-kappaB (nuclear factor kappaB) reporter assays demonstrated that co-expression of either full-length CLAN or the NACHT domain of CLAN significantly inhibited NF-kappaB activation induced by Nod1 or Nod2 overexpression. In addition, co-expression of CLAN or the NACHT domain of CLAN with Nod1 or Nod2 inhibited the ability of these proteins to generate active IL-1beta (interleukin 1beta) through their association with pro-caspase 1. The NACHT domain of CLAN was demonstrated by co-immunoprecipitation experiments to bind all NACHT domains that were tested, including the NACHT domains from CLAN itself, Nod1, Nod2, cryopyrin, NAC, PAN2 [PAAD [pyrin, AIM (absent-in-melanoma), ASC (apoptosis-associated speck-like protein containing a CARD) and death-domain-like]- and NACHT-containing protein] and NAIP (neuronal apoptosis inhibitory protein). Finally, monocyte-expressed CLAN was found to associate with Nod2 following exposure to bacterial peptidoglycan, implying a regulatory role for interaction of these NACHT proteins in the innate immune response. These studies suggest that by mediating hetero-oligomerization, NACHT domains provide a means by which various NACHT-containing proteins may interact, creating protein-interaction networks that potentially modulate immune responses to invading pathogens.

Figure 1. Interaction of NACHT-family proteins

HEK-293T cells were transfected with epitope-tagged expression plasmids encoding the complete coding sequences for various proteins as indicated. Co-immunoprecipitations (IP) were carried out using anti-FLAG- or anti-myc-conjugated agarose beads and immune complexes were analysed by SDS/PAGE and immunoblotting (WB) using antibodies that detect myc or FLAG epitopes. As a control, some lysates were subjected to IP with normal mouse IgG (left-hand panel, lane 2). Expression of proteins was verified by analysing 10% of each lysate taken before immunoprecipitation.

Figure 2. Heterotypic NACHT-domain interactions

HEK-293T cells were transfected with epitope-tagged expression plasmids encoding various NACHT domains or other proteins as indicated. Co-immunoprecipitations (IP) were performed using anti-FLAG-conjugated agarose beads and immune complexes were analysed using by SDS/PAGE and immunoblotting (WB). As a control, some samples were subjected to immunoprecipitation with normal mouse IgG (lane 2). Expression of proteins was verified by analysing 10% of each lysate taken before immunoprecipitation.

Figure 3. Gel filtration analysis of the NACHT domain of CLAN

FLAG-tagged CLAN-NACHT was transiently expressed in HEK-293T cells, followed by lysis in 100 mM NaCl, 0.1% Nonidet P40, 1 mM EDTA and 1 mM dithiothreitol. Extracts were separated on a Superdex-200 HR column, and fractions were collected and subjected to immunoprecipitation with anti-FLAG–agarose conjugate. The blot is representative of two independent experiments.

Figure 4. CLAN inhibits NF-κB activation induced by Nod1 and Nod2

(A) Domain architecture and amino acid composition of the CLAN deletion mutants used. (B, upper panel) HEK-293T cells were seeded into 24-well plates and transfected the following day with 100 ng of p-NF-κB-luc and 50 ng of pTK-RL reporter gene plasmids together with 100 ng plasmids encoding Nod1 or Nod2 and either 100 ng or 1 μg of plasmid encoding CLAN. Cells were lysed 24 h later and luciferase activity was determined using an automated luminometer. Results are means±S.D. of fold induction of NF-κB activity relative to control-transfected cells (n=3) after normalization for transfection efficiency based on Renilla luciferase activity. *P≤0.001, determined by one-way ANOVA and Bonferroni's comparison test, comparing Nod1 or Nod2 alone versus in combination with CLAN. (B, lower panel) Lysates from equal numbers of transfected cells were analysed by SDS/PAGE and immunoblotting, using anti-myc antibody with ECL®-based detection. Densitometry analysis indicates that the ratio of expression of CLAN(10X)/Nod2 is approx. 4:1. (C, upper panel) HEK-293T cells were transfected with Nod2 (50 ng) in conjunction with CLAN (800 ng), CLAN[NACHT] (400 ng) or Apaf-1[NB-ARC] (600 ng), as indicated. (C, lower panel) Lysates from equal numbers of transfected cells were analysed by SDS/PAGE and immunoblotting, using anti-myc antibody with ECL®-based detection, to compare the levels of expression of myc-Nod2, myc-CLAN, myc-CLAN[NACHT] or myc-Apaf-1[NB-ARC]. Densitometry analysis indicates that the ratio of expression of CLAN(10X)/Nod2 and CLAN[NACHT]/Nod2 is approx. 4:1. Results are representative of three independent experiments.

Figure 5. CLAN-mediated suppression of Nod2-induced NF-κB is specific and requires only the NACHT domain

The indicated expression plasmids were co-transfected into HEK-293T (Nod2, 50 ng; others, 400 ng) with a luciferase reporter plasmid and cells were lysed 24 h later. Luciferase activity was determined using an automated luminometer. Results are means±S.D. (n=3) expressed as fold activation relative to unstimulated cells transfected with pcDNA3 (=1.0). *P<0.001 relative to Nod2 alone.

Figure 6. CARD-carrying NACHT-family proteins bind pro-caspase 1

HEK-293T cells were transfected with epitope-tagged expression plasmids encoding various full-length NACHT-family proteins or caspases, as indicated. Co-immunoprecipitations (IP) were performed using anti-FLAG-conjugated agarose beads and immune complexes were analysed using SDS/PAGE and immunoblotting (WB). As a control, some samples were subjected to immunoprecipitation with normal mouse IgG (lane 2). Expression of proteins was verified by analysing 10% of each lysate taken before immunoprecipitation.

Figure 7. CLAN inhibits caspase 1 activation induced by Nod2

(A) HEK-293T cells were transfected with plasmids encoding pro-IL-1β (400 ng) and caspase 1 (100 ng), with (+) or without (−) Nod2 (50 ng) or CLAN [+, 50 ng; ++, 500 ng]. Supernatants were analysed for IL-1β secretion by ELISA at 24 h post-transfection. *P<0.05, relative to Nod2 alone. (B) HEK-293T cells were transfected with plasmids encoding pro-IL-1β (400 ng) and caspase 1 (100 ng), with (+) or without (−) Nod2 (50 ng), CLAN (400 ng), CLAN[NACHT] (400 ng), Apaf-1[NB-ARC] (400 ng) or caspase 4 (100 ng), as indicated. Supernatants were analysed for IL-1β secretion by ELISA at 24 h post-transfection. (C) HEK-293T cells were transfected with plasmids encoding pro-IL-1β (400 ng) and caspase 1 (100 ng), with (+) or without (−) CLAN[ΔLRR] (100 ng [+] or 500 ng [++]), Nod2[ΔLRR] (100 ng), CLAN[NACHT] (500 ng) or ASC (200 ng). Supernatants were analysed for IL-1β secretion by ELISA 24 h post-transfection. *, Experimental conditions that resulted in significantly less IL-1β secretion compared with Nod2 alone (P<0.05). Results are means±S.D. representative of at least two independent experiments.

Figure 8. CLAN and Nod2 co-associate in monocytes following exposure to PGN

THP-1 cells expressing epitope-tagged CLAN (THP-1/CLAN) or neomycin-resistant control cells (THP-1/Neo) were exposed to PGN (10 μg/ml) for the times indicated, then lysed, and subjected to anti-myc immunoprecipitation and immunoblotting for detection of associated Nod2 protein. To assess equivalent immunoprecipitation of CLAN, membranes were stripped and re-probed with anti-myc antibodies. As a control, some samples were subjected to immunoprecipitation with normal mouse IgG (lanes 1 and 4). The blot is representative of three independent experiments.