SummaryThe rhodanese homology domain is a ubiquitous fold found in several phylogenetically related proteins encoded by eubacterial, archeal, and eukaryotic genomes. Although rhodanese-like proteins share evolutionary relationships, analysis of their sequences highlights that they are so heterogeneous to form the rhodanese superfamily. The variability occurs at different levels including sequence, active site loop length, presence of a critical catalytic Cys residue, and domain arrangement. Even within the same genome, multiple genes encode rhodanese-like proteins presenting with variably arranged rhodanese domain(s): as single or tandem domain(s), or combined with other protein domain(s). Given the highly variable organization of the rhodanese domain(s) and the context where it is found, here we review the structural organization and function of the rhodanese-like proteins. The overview of the most recent findings about rhodanese allow us to depict a superfamily of versatile proteins relying on persulfide chemistry to accomplish cellular functions spanning from resistance to environmental threats, such as cyanide, and key cellular reactions related to sulfur metabolism and progression of cell cycle. IUBMB Life, 59: 51-59, 2007

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem–human serum albumin (ferric HSA–haem) and of ferrous nitrosylated haem–human serum albumin (ferrous HSA–haem‐NO) is reported. Ferric HSA–haem is hexa‐coordinated, the haem‐iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules − buried in close proximity to the haem binding pocket − induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem‐iron, and the quenching of the 1H‐NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA–haem, corresponding to the ibuprofen secondary cleft, are 5.4 ± 1.1 × 10−4 m and 2.1 ± 0.4 × 10−5 m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA–haem is lower than that reported for drug binding to haem‐free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 ± 0.2 × 10−8 m in the absence of drugs to 1.5 ± 0.2 × 10−7 m in the presence of ibuprofen and warfarin. Ferrous HSA–haem‐NO is a five‐coordinated haem‐iron system. Drug binding to the warfarin primary site of ferrous HSA–haem‐NO induces the transition towards the six‐coordinated haem‐iron species, the haem‐iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear‐cut evidence for the drug‐induced shift of allosteric equilibrium(a) of HSA.

List of abbreviations: K, keratin; p63-/-, mouse knockout for p63; p63-/-;TA, p63 knockout mice complemented with TAp63; p63-/-;ΔN, p63 knockout mice complemented with ΔNp63; p63-/-;TA;ΔN, p63 knockout mice complemented with both TAp63 and ΔNp63;IKKα, IkB kinase-α; TA, transactivation domain; ΔN, amino-terminal truncated protein. AbstractThe epidermis, the outer layer of the skin composed of keratinocytes, is a stratified epithelium that functions as a barrier to protect the organism from dehydration and external insults. The epidermis develops following the action of the transcription factor p63, a member of the p53 family of transcription factors. The Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal transactivation domain (TAp63) and one without (DeltaNp63), although their relative contribution to epidermal development is not clearly established. Trp63 mutations are involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. In this review we summarise the current advances that have been made in understanding the role of p63 in epidermal morphogenesis.

SummaryHeterozygous mutations of p63, a key transcription factor in epithelial development, are causative in a variety of human ectodermal dysplasia disorders. Although the mutation spectrum of these disorders displays a striking genotype-phenotype association, the molecular basis for this association is only superficially known. Here, we characterize the transcriptional activity and protein stability of Np63 mutants (that is, mutants of a p63 isoform that lacks the N-terminal transactivation domain) that are found in ectrodactylyectodermal dysplasia-cleft syndrome (EEC), ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC) and nonsyndromic split-hand/split-foot malformation (SHFM). DNA-binding and sterile alpha motif (SAM) domain mutants accumulate in the skin of EEC and AEC syndrome patients, respectively, and show extended half lives in vitro. By contrast, C-terminal mutations found in SHFM patients have half-lives similar to that of the wild-type protein. The increased half-life of EEC and AEC mutant proteins was reverted by overexpression of wild-type Np63. Interestingly, the mutant proteins exhibit normal binding to and degradation by the E3 ubiquitin ligase Itch. Finally, EEC and AEC mutant proteins have reduced transcriptional activity on several skin-specific gene promoters, whereas SHFM mutant proteins are transcriptionally active. Our results, therefore, provide evidence for a regulatory feedback mechanism for p63 that links transcriptional activity to regulation of protein homeostasis by an unknown mechanism. Disruption of this regulatory mechanism might contribute to the pathology of p63-related developmental disorders.