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Interleukin-1? Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin.

ABSTRACT: The secretion of IL-1? is a central event in the initiation of inflammation. Unlike most other cytokines, the secretion of IL-1? requires two signals: one signal to induce the intracellular up-regulation of pro-IL-1? and a second signal to drive secretion of the bioactive molecule. The release of pro-IL-1? is a complex process involving proteolytic cleavage by caspase-1. However, the exact mechanism of secretion is poorly understood. Here we sought to identify novel proteins involved in IL-1? secretion and intracellular processing to gain further insights into the mechanism of IL-1 release. A human proteome microarray containing 19,951 unique proteins was used to identify proteins that bind human recombinant pro-IL-1?. Probes with a signal-to-noise ratio of >3 were defined as biologically relevant. In these analyses, calmodulin was identified as a particularly strong hit, with a signal-to-noise ratio of ? 11. Using an ELISA-based protein-binding assay, the interaction of recombinant calmodulin with pro-IL-1?, but not mature IL-1?, was confirmed and shown to be calcium-dependent. Finally, using small molecule inhibitors, it was demonstrated that both calcium and calmodulin were required for nigericin-induced IL-1? secretion in THP-1 cells and primary human monocytes. Together, these data suggest that, following calcium influx into the cell, pro-IL-1? interacts with calmodulin and that this interaction is important for IL-1? processing and release.

SUBMITTER: Ainscough JS

PROVIDER: S-EPMC4692238 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Interleukin-1β Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin.

Ainscough Joseph S JS Gerberick G Frank GF Kimber Ian I Dearman Rebecca J RJ

The Journal of biological chemistry 20151111 52

The secretion of IL-1β is a central event in the initiation of inflammation. Unlike most other cytokines, the secretion of IL-1β requires two signals: one signal to induce the intracellular up-regulation of pro-IL-1β and a second signal to drive secretion of the bioactive molecule. The release of pro-IL-1β is a complex process involving proteolytic cleavage by caspase-1. However, the exact mechanism of secretion is poorly understood. Here we sought to identify novel proteins involved in IL-1β se ...[more]

PMID: 26559977

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Project description:Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter. Keywords: strain_or_line_design

Dataset Information

Interleukin-1? Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin.

Publications

Interleukin-1β Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin.

Similar Datasets

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