Transport of iron across the cell membrane is a tightly controlled process carried out by specific proteins in all living cells. In yeast and in mammals, a system formed by an enzyme with ferroxidase activity coupled to a membrane transporter supports iron uptake or iron efflux, respectively. Ferroxidase belongs to the family of blue multicopper oxidases, enzymes able to couple the one-electron oxidation of substrate(s) to full reduction of molecular oxygen to water. On the other hand, the permeases are widely different and are specific to Fe³⁺ and Fe²⁺ in yeast and multicellular organisms, respectively. This review will describe the yeast and human ferroxidase–permease systems, highlighting similarities and differences in structure, function and regulation of the respective protein components.

Histone acetyltransferases (HATs) are involved in the epigenetic positive control of gene expression in eukaryotes. CREB-binding proteins (CBP)/p300, a subfamily of highly conserved HATs, have been shown to function as acetylases on both histones and non-histone proteins. In the model plant Arabidopsis thaliana among the five CBP/p300 HATs, HAC1, HAC5 and HAC12 have been shown to be involved in the ethylene signaling pathway. In addition, HAC1 and HAC5 interact and cooperate with the Mediator complex, as in humans. Therefore, it is potentially difficult to discriminate the effect on plant development of the enzymatic activity with respect to their Mediator-related function. Taking advantage of the homology of the human HAC catalytic domain with that of the Arabidopsis, we set-up a phenotypic assay based on the hypocotyl length of Arabidopsis dark-grown seedlings to evaluate the effects of a compound previously described as human p300/CBP inhibitor, and to screen previously described cinnamoyl derivatives as well as newly synthesized analogues. We selected the most effective compounds, and we demonstrated their efficacy at phenotypic and molecular level. The in vitro inhibition of the enzymatic activity proved the specificity of the inhibitor on the catalytic domain of HAC1, thus substantiating this strategy as a useful tool in plant epigenetic studies.

Ferroportin (Fpn), a member of the major facilitator superfamily (MFS) of transporters, is the only known iron exporter found in mammals and plays a crucial role in regulating cellular and systemic iron levels. MFSs take on different conformational states during the transport cycle: inward open, occluded, and outward open. However, the precise molecular mechanism of iron translocation by Fpn remains unclear, with conflicting data proposing different models. In this work, amber codon suppression was employed to introduce dansylalanine (DA), an environment-sensitive fluorescent amino acid, into specific positions of human Fpn (V46, Y54, V161, Y331) predicted to undergo major conformational changes during metal translocation. The results obtained indicate that different mutants exhibit distinct fluorescence spectra depending on the position of the fluorophore within the Fpn structure, suggesting that different local environments can be probed. Cobalt titration experiments revealed fluorescence quenching and blue-shifts of λ_max in Y54DA, V161DA, and Y331DA, while V46DA exhibited increased fluorescence and blue-shift of λ_max. These observations suggest metal-induced conformational transitions, interpreted in terms of shifts from an outward-open to an occluded conformation. Our study highlights the potential of genetically incorporating DA into Fpn, enabling the investigation of conformational changes using fluorescence spectroscopy. This approach holds great promise for the study of the alternating access mechanism of Fpn and advancing our understanding of the molecular basis of iron transport.

Local disruption of iron homeostasis leading to oxidative stress is considered one of the main mechanisms of asbestos-dependent cyto-/genotoxicity. Another aspect contributing to the risk of developing pathological consequences upon asbestos exposure is due to individual genetic fac-tors. In a previous study we identified a coding SNP in the hephaestin gene (HEPH) exerting a protective activity against the development of asbestos related thoracic cancers. Heph is a fer-roxidase which promotes iron export in concert with the permease ferroportin (Fpn1). Here, we performed an in-depth functional characterization of the HephD568H variant to gain insights on the molecular basis of its protective activity in asbestos-related carcinogenesis. We demonstrat-ed that HephD568H complexes with Fpn1 and possesses a full ferroxidase activity. Despite being more efficiently recruited at the plasma membrane, HephD568H is severely hampered in (iron-depleted) apo-Tfn binding, whose interaction with the WT ferroxidase emerged as a novel mechanism to perceive brain iron needs. Heph in human lung is expressed by pericytes and fi-broblasts and lung pericytes were shown to respond to iron request by up-regulating the iron exporter pair. These findings extend to the pulmonary vascular unit the paradigm of local iron regulation uncovered at the blood-brain barrier. Moreover, they mechanistically correlate alter-ations in iron sensing with the risk of developing asbestos-dependent malignancies.

A local disruption of iron homeostasis leading to oxidative stress is considered one of the main mechanisms of asbestos-related genotoxicity. Another aspect contributing to the risk of developing pathological consequences upon asbestos exposure is individual genetic factors. In a previous study, we identified a coding SNP in the hephaestin gene (HEPH) that protects against developing asbestos-related thoracic cancer. Heph is a ferroxidase that promotes iron export in concert with the permease ferroportin (Fpn1). Here, we performed an in-depth functional characterization of the HephD568H variant to gain insights into the molecular basis of its protective activity. We showed that HephD568H forms a complex with Fpn1 and possesses full ferroxidase activity. Although HephD568H is more efficiently recruited to the plasma membrane, it is impaired in binding iron-deficient Tfn, whose interaction with wild-type (WT) ferroxidase emerged as a novel mechanism to perceive brain iron needs. Heph is expressed in the human lung by pericytes and fibroblasts, and lung pericytes were shown to respond to iron demand by upregulating the iron exporter pair. These results extend the paradigm of local iron regulation discovered at the blood–brain barrier to the pulmonary vasculature. Furthermore, they establish a mechanistic link between changes in iron sensing and the risk of developing asbestos-related malignancies.