Nod1, a CARD protein, enhances pro-interleukin-1β processing through the interaction with pro-caspase-1

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Abstract

The production of bioactive interleukin-1β (IL-1β), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1β processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1β secretion, as well as lipopolysaccharide (LPS)-induced IL-1β secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1β secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1β secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.

Section snippets

Materials and methods

Plasmids. The expression plasmids, pcDNA3-Myc-caspase-1 (wild type), pcDNA3-caspase-1-Myc (wild type), pcDNA3-Myc-caspase-1 (Cys 285 Ser), pcDNA3-HA-caspase-1 (Cys 285 Ser), pcDNA3-Flag-RIP2, and pCMV-IL-1β, have been described previously [21]. pcDNA3-Nod1-Flag, pcDNA3-Flag-Nod1-CARD (residues 1–158), pcDNA3-Myc-RIP2-CARD (residues 365–531), pcDNA3-Flag-ICEBERG, and pcDNA3-Myc-Nod1 were created by polymerase chain reaction (PCR).

Co-immunoprecipitations and immunoblotting assays. Human embryonic

Nod1 binds to both caspase-1 and RIP2 through CARD–CARD interaction

Because an earlier report described that Nod1 interacts both with pro-caspase-1 and RIP2 [13], we tested the possibility that these interactions are mediated through heterotypic CARD–CARD interactions. For these experiments, 293T cells were transiently transfected with expression plasmids encoding Flag-tagged-Nod1-CARD either with an expression plasmid producing HA-tagged caspase-1-CARD or Myc-tagged RIP2-CARD protein. Immunoprecipitations were then performed with anti-Flag antibody and the

Discussion

It has been known that pro-caspase-1 interacts with Nod1 [13]. We therefore undertook a study of the consequences of this interaction. Because the key role of caspase-1 is IL-1β processing, we investigated whether Nod1 influences IL-1β secretion induced by the expression of caspase-1. Nod1 interacts with caspase-1 through CARD–CARD interactions, thereby promoting caspase-1 oligomerization (Fig. 1). Consequently, Nod1 expression increases caspase-1-induced secretion of mature IL-1β in 293T

Acknowledgements

This work was supported by the KOSEF through the Cell death Disease Center at the Catholic University of Korea.

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    Abbreviations: IL-1β, interleukin-1β; CARD, caspase recruit domain; NOD, nucleotide-binding oligomerization domain; PCR, polymerase chain reaction; DAPI, 4,6-diamidine-2-phenylindole dihydrochloride; GFP, green fluorescent protein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; LPS, lipopolysaccharide; LRR, leucine-rich repeat.

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