The ZIP6/ZIP10 heteromer is essential for the zinc-mediated trigger of mitosis

Zinc has been known to be essential for cell division for over 40 years but the molecular pathways involved remain elusive. Cellular zinc import across biological membranes necessitates the help of zinc transporters such as the SLC39A family of ZIP transporters. We have discovered a molecular process that explains why zinc is required for cell division, involving two highly regulated zinc transporters, as a heteromer of ZIP6 and ZIP10, providing the means of cellular zinc entry at a specific time of the cell cycle that initiates a pathway resulting in the onset of mitosis. Crucially, when the zinc influx across this heteromer is blocked by ZIP6 or ZIP10 specific antibodies, there is no evidence of mitosis, confirming the requirement for zinc influx as a trigger of mitosis. The zinc that influxes into cells to trigger mitosis additionally changes the phosphorylation state of STAT3 converting it from a transcription factor to a protein that complexes with this heteromer and pS38Stathmin, the form allowing microtubule rearrangement as required in mitosis. This discovery now explains the specific cellular role of ZIP6 and ZIP10 and how they have special importance in the mitosis process compared to other ZIP transporter family members. This finding offers new therapeutic opportunities for inhibition of cell division in the many proliferative diseases that exist, such as cancer

CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin

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

DNA sequence variation and methylation in an arsenic tolerant earthworm population

Evidence is emerging that earthworms can evolve tolerance to trace element enriched soils. However, few studies have sought to establish whether such tolerance is determined through adaption or plasticity. Here we report results from a combined analysis of mitochondrial (cytochrome oxidase II, COII), nuclear (amplified fragment length polymorphism, AFLP) variation and DNA methylation in populations of the earthworm Lumbricus rubellus from sites across an abandoned arsenic and copper mine. Earthworms from the mine site population demonstrated clear arsenic tolerance in comparison to a naïve strain. COII and AFLP results suggest that L. rubellus from the unexposed and the adapted populations comprises two cryptic lineages (Linages A and B) each of which was present across all of the sites. AFLP analysis by lineage highlighted variations associated with soil metal/metalloid concentrations (most clearly for Lineage A) suggesting a genetic component to the observed tolerance. The methylation sensitive AFLP (Me-AFLP) identified a high genome methylation content (average 13.5%) in both lineages. For Lineage A, Me-AFLP analysis did not identify a strong association with soil arsenic levels. For Lineage B, however, a clear association of methylation patterns with soil arsenic concentrations was found. This suggests that Lineage B earthworms utilise epigenetic mechanisms to adapt to the presence of contamination. These fundamentally different genetic adjustments in the two clades indicate that the two lineages employ distinct adaptive strategies (genetic or epigenetic) in response to arsenic exposure. Mechanisms driving this variation may be founded within the colonisation histories of the lineages

CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin

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

Aryl hydrocarbon receptor utilises cellular zinc signals to maintain the gut epithelial barrier

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