PIR2/Rnf144B regulates epithelial homeostasis by mediating degradation of p21WAF1 and p63.

ΔNp63 is a transcription factor that is critical for the development of stratified epithelia and is overexpressed or amplified in >80% of squamous cell carcinomas (SCCs). We identified the RING finger E3 ubiquitin ligase PIR2/Rnf144b as a direct transcriptional target of ΔNp63α and showed that its expression parallels that of ΔNp63α in keratinocytes, SCC cell lines and SCCs. We used primary keratinocytes as a model system to investigate the function of PIR2/Rnf144b in stratified epithelia. Depletion of PIR2/Rnf144b severely impaired keratinocyte proliferation and differentiation, associated with accumulation of p21(WAF1/CIP1); a known target of PIR2/Rnf144b. More importantly, we found that PIR2/Rnf144b binds and mediates proteasomal degradation of ΔNp63α, generating a hitherto unknown auto-regulatory feedback loop. These findings substantiate PIR2/Rnf144b as a potentially critical component of epithelial homeostasis, acting downstream of ΔNp63α to regulate cellular levels of p21(WAF1/CIP1) and ΔNp63α.

Insights into the role of the histidines in the structure and stability of cytochrome c.

In this paper we investigate the role played by each histidine in the amino acid sequence of yeast iso-1-cytochrome c (with the exception of H18, the residue axially coordinated to the heme iron) in determining the protein structure and stability. To this end, we have generated and characterized the double mutants H26Y/H33Y, H26Y/H39K and H33Y/H39K obtained from the C102T variant of the protein, which retain only one histidine side chain in the amino acid sequence. In particular, the H39K mutation inserts a lysine at position 39 as in the sequence of equine cytochrome c. The H26Y/H33Y/H39K triple mutant, which lacks all three histidines, was also produced and its spectroscopic properties are compared with those of the double mutants. The data highlight the critical role played by H26 in determining protein stability. Recombinant horse cytochrome c and the corresponding H26Y mutant were also generated and characterized. Since equine cytochrome c exhibits higher stability than the yeast protein, this provides a valuable opportunity to understand the role played by the invariant H26 residue in determining structure and stability.

ATP specifically drives refolding of non-native conformations of cytochrome c.

An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.

Rupture of the hydrogen bond linking two Omega-loops induces the molten globule state at neutral pH in cytochrome c.

His26Tyr and His33Tyr mutants were obtained from the Cys102Thr variant of yeast iso-1-cytochrome c. Spectroscopic studies show that a mutation at position 26 at pH 7.0 enhances flexibility of the peptide, alters the heme pocket region and the axial coordination to heme-iron, and reduces protein stability. The His26Tyr mutant shows properties typical of the molten globule. Further, formation of an axially misligated minor low spin species occurs with partial displacement of Met80, the axial ligand of the heme-iron in the native protein. The pK(a) determined for the alkaline transition of this mutant is 7.48 (+/- 0.05), approximately 0.5 lower than that of the wild-type protein. Hence, the alkaline conformer is populated at pH 7.0, and the sixth ligand of the misligated species is proposed to be a lysine. Furthermore, a reduction in catalytic activity indicates that the functional properties are altered. The results suggest that the structural and functional changes observed in the His26Tyr mutant are because the mutation frees the two Omega-loops that, in the native protein, are linked by the hydrogen bond between His26 and Glu44. Hence, one may infer that the His26-Glu44 hydrogen bond is essential for the rigidity and stability of the native protein. In its absence, the heightened flexibility of the peptide fold results in conversion of the macromolecule to a molten globule state, even at neutral pH. Ligand exchange at the sixth coordination position of the heme-iron(III) observed as the minor species (i.e., the alkaline conformer) is therefore induced by a long-range effect. This result is of interest since mutations reported to date, which stabilize the alkaline conformer, all occur in the loop including Met80. By contrast, only very minor spectroscopic (and, thus, structural) changes are observed for the His33Tyr mutant. This suggests that His33 does not form intramolecular bonds considered important for the protein structure and stability, and is consistent with the high variability of residues at position 33 in cytochromes c.