Functional genomics of drug-induced ion homeostasis identifies a novel regulatory crosstalk of iron and zinc regulons in yeast.

Pyrrolidine dithiocarbamate (PDTC), a known inhibitor of NFκB activation, has antioxidative as well as antiviral activities. PDTC is effective against several virus families, indicating that its antiviral mechanism targets host rather than viral functions. To investigate its mode of action, we used baker's yeast as a simple eukaryotic model system and two types of genome-wide analysis. First, expression profiling using whole-genome DNA microarrays identifies more than 200 genes differentially regulated upon PDTC exposure. Interestingly, the Aft1-dependent iron regulon is a main target of PDTC, indicating a lack of iron availability. Moreover, the PDTC-caused zinc influx triggers a strong regulatory effect on zinc transporters due to the cytoplasmic zinc excess. Second, phenotypic screening the EUROSCARF collection for PDTC hypersensitivity identifies numerous mutants implicated in vacuolar maintenance, acidification as well as in transport, mitochondrial organization, and translation. Notably, the screening data indicate significant overlaps of PDTC-sensitive genes and those mediating zinc tolerance. Hence, we show that PDTC induces cytoplasmic zinc excess, eliciting vacuolar detoxification, which in turn, disturbs iron homeostasis and activates the iron-dependent regulator Aft1. Our work reveals a complex crosstalk in yeast ion homeostasis and the underlying regulatory networks.

A mutation in the first ligand-binding repeat of the human very-low-density lipoprotein receptor results in high-affinity binding of the single V1 module to human rhinovirus 2.

Minor group human rhinoviruses (HRVs) bind members of the low-density lipoprotein receptor family for cell entry. The ligand-binding domains of these membrane proteins are composed of various numbers of direct repeats of about 40 amino acids in length. Residues involved in binding of module 3 (V3) of the very-low-density lipoprotein receptor (VLDLR) to HRV2 have been identified by X-ray crystallography (N. Verdaguer, I. Fita, M. Reithmayer, R. Moser, and D. Blaas, Nat. Struct. Mol. Biol. 11:429-434, 2004). Sequence comparisons of the eight repeats of VLDLR with respect to the residues implicated in the interaction between V3 and HRV2 suggested that (in addition to V3) V1, V2, V5, and V6 also fulfill the requirements for interacting with the virus. Using a highly sensitive binding assay employing phage display, we demonstrate that single modules V2, V3, and V5 indeed bind HRV2. However, V1 does not. A single mutation from threonine 17 to proline converted the nonbinding wild-type form of V1 into a very strong binder. We interpret the dramatic increase in affinity by the generation of a hydrophobic patch between virus and receptor; in the presence of threonine, the contact area might be disturbed. This demonstrates that the interaction between virus and its natural receptors can be strongly enhanced by mutation.