Search

Abstract Zinc and plant-derived ligands of the aryl hydrocarbon receptor (AHR) are dietary components affecting intestinal epithelial barrier function. Here, we explore whether zinc and the AHR pathway are linked. We show that dietary supplementation with an AHR pre-ligand offers protection against inflammatory bowel disease in a mouse model while protection fails in mice lacking AHR in the intestinal epithelium. AHR agonist treatment is also ineffective in mice fed zinc depleted diet. In human ileum organoids and Caco-2 cells, AHR activation increases total cellular zinc and cytosolic free Zn2+ concentrations through transcription of genes for zinc importers. Tight junction proteins are upregulated through zinc inhibition of nuclear factor kappa-light-chain-enhancer and calpain activity. Our data show that AHR activation by plant-derived dietary ligands improves gut barrier function at least partly via zinc-dependent cellular pathways, suggesting that combined dietary supplementation with AHR ligands and zinc might be effective in preventing inflammatory gut disorders.

Hemocyanins are multimeric oxygen transport proteins present in the blood of arthropods and molluscs, containing up to 8 oxygen-binding functional units per monomer. In molluscs, hemocyanins are assembled in decamer ‘building blocks’ formed of 5 dimer ‘plates’, routinely forming didecamer or higher-order assemblies with d5 or c5 symmetry. Here we describe the cryoEM structures of the didecamer (20-mer) and tridecamer (30-mer) forms of a novel hemocyanin from the slipper limpet Crepidula fornicata (SLH) at 7.0 and 4.7 Å resolution respectively. We show that two decamers assemble in a ‘tail-tail’ configuration, forming a partially capped cylinder, with an additional decamer adding on in ‘head-tail’ configuration to make the tridecamer. Analysis of SLH samples shows substantial heterogeneity, suggesting the presence of many higher-order multimers including tetra- and pentadecamers, formed by successive addition of decamers in head-tail configuration. Retrieval of sequence data for a full-length isoform of SLH enabled the use of Alphafold to produce a molecular model of SLH, which indicated the formation of dimer slabs with high similarity to those found in keyhole limpet hemocyanin. The fit of the molecular model to the cryoEM density was excellent, showing an overall structure where the final two functional units of the subunit (FU-g and FU-h) form the partial cap at one end of the decamer, and permitting analysis of the subunit interfaces governing the assembly of tail-tail and head-tail decamer interactions as well as potential sites for N-glycosylation. Our work contributes to the understanding of higher-order oligomer formation in molluscan hemocyanins and demonstrates the utility of Alphafold for building accurate structural models of large oligomeric proteins.