Structural model of dodecameric heat-shock protein Hsp21:Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations

Impurities in technical mixtures of chlorinated paraffins show AhR agonist properties as determined by the DR-CALUX bioassay

Chlorinated paraffins (CPs) are produced at more than one million tons per year. Technical CPs mixtures may contain impurities, which end up in consumer products. In the present study, 17 technical CPs mixtures were investigated for the potential occurrence of potential impurities. By applying the DR-CALUX bioassay, 3 out of 17 technical mixtures were shown to elicit responses at 4 h exposure time, but much lower at 48 h. Constitutional defined CPs materials did not show responses. Subsequently different groups of known AhR-agonists and compounds suspected to be present in technical CPs mixtures were investigated. Benzene, (poly)chlorobenzene, non-dioxin like polychlorinated naphthalenes (PCNs), and three-ringed polyaromatic hydrocarbons (PAHs) did not result in a significant response at 4 h or 48 h. TCDD, non-ortho PCBs, dioxin-like PCNs, four or five ringed PAHs and their chlorinated analogues resulted in a significant response. TCDD and the non-ortho PCBs showed the highest potency and stability, while dioxin-like PCNs, PAHs, and the chlorinated PAHs were clearly inactivated (metabolized) at longer incubation. Altogether, the present findings substantiate that AhR-mediated responses of CPs technical mixtures in the DR-CALUX bioassay are caused by impurities, most likely some intermediate stable AhR-agonists such as dioxin-like PCNs or (chlorinated) PAHs. The current study shows that impurities in CPs technical mixtures need to be investigated for assessing the safety of technical CPs mixtures.</p

Presence and risks of polycyclic aromatic hydrocarbons, dioxins and dioxin-like PCBs in dietary plant supplements as elucidated by a combined DR CALUX® bioassay and GC-HRMS based approach

Plant-based dietary supplements may contain undesirable contaminants such as polycyclic aromatic hydrocarbons, dioxins and dioxin-like polychlorinated biphenyls (dl-PCBs) due to the sources of raw materials or processing methods used. The presence of these contaminants in a series of herbal supplements sold on the Ghanaian market for improving sexual performance was examined using the DR CALUX® bioassay in combination with GC-HRMS analysis. Overall, cell responses at 4 and 48 h exposure to extracts prepared without an acid-silica clean-up were relatively higher than the responses obtained from extracts prepared with an acid-silica clean-up. This indicated that the 40 supplements contained only low levels of stable aryl hydrocarbon receptor (AhR) agonists like polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dl-PCBs, while some contained substantial amounts of less stable AhR-agonists. Ten supplements selected for confirmation with GC-HRMS analysis contained PCDD/Fs and dl-PCBs at levels ranging from 0.01 to 0.19 pg toxic equivalent (TEQ)/g only, while the level of the sum of 4 polycyclic aromatic hydrocarbons (Σ4PAHs) representing less stable AhR agonists, ranged from not detected (ND) to 25.5 ng/g. These concentrations were in line with the responses observed in the DR CALUX® bioassay. The concentration of PCDD/Fs and dl-PCBs corresponded to estimated daily intakes (EDIs) ranging from 0.01 to 1.20 pg TEQ/day, or 0.001 to 0.12 pg TEQ/kg bw/week for a 70 kg bw consumer, which was below the established tolerable weekly intake (TWI) of 2 pg TEQ/kg bw/week, thus indicating low concern for consumers’ health. Similarly, the EDIs based on the detected Σ4PAHs in supplements ranged from 7.2 to 111 ng/day, or 0.1 to 1.6 ng/kg bw/day, which corresponded to MOE values above 10,000, indicating a low health concern