Maria Cristina Piro

Extended cardiolipin anchorage to cytochrome c: a model for protein-mitochondrial membrane binding.

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.

Differential control of TAp73 and ΔNp73 protein stability by the ring finger ubiquitin ligase PIR2

p73 is a p53-related transcription factor with fundamental roles in development and tumor suppression. Transcription from two different promoters on the p73 gene results in generation of transcriptionally active TAp73 isoforms and dominant negative ΔNp73 isoforms with opposing pro- and anti-apoptotic functions. Therefore, the relative ratio of each isoform is an important determinant of the cell fate. Proteasomal degradation of p73 is mediated by polyubiquitination-dependent and -independent processes both of which appear, thus far, to lack selectivity for the TAp73 and ΔNp73 isoforms. Here, we describe the characterization of another transcriptional target of TAp73; a ring finger domain ubiquitin ligase p73 Induced RING 2 protein (PIR2). Although PIR2 was initially identified a p53-induced gene (p53RFP), low abundance of PIR2 transcript in mouse embryonic fibroblasts of TAp73 KO mice compared with WT mice and comparison of PIR2 mRNA and protein levels following TAp73 or p53 overexpression substantiate TAp73 isoforms as strong inducers of PIR2. Although PIR2 expression was induced by DNA damage, its expression did not alter apoptotic response or cell cycle profile per se. However, coexpression of PIR2 with TAp73 or ΔNp73 resulted in an increase of the TA/ΔNp73 ratio, due to preferential degradation of ΔNp73. Finally, PIR2 was able to relieve the inhibitory effect of ΔNp73 on TAp73 induced apoptosis following DNA damage. These results suggest that PIR2, by being induced by TAp73 and degrading ΔNp73, differentially regulates TAp73/ΔNp73 stability, and, hence, it may offer a therapeutic approach to enhance the chemosensitivity of tumor cells.

ATP Acts as a Regulatory Effector in Modulating Structural Transitions of Cytochrome c: Implications for Apoptotic Activity†

The binding of lipids (free fatty acids as well as acidic phospholipids) to cytochrome c (cyt c) induces conformational changes and partial unfolding of the protein, strongly influencing cyt c oxidase/peroxidase activity. ATP is unique among the nucleotides in being able to turn non-native states of cyt c back to the native conformation. The peroxidase activity acquired by lipid-bound cyt c turns out to be very critical in the early stages of apoptosis. Nucleotide specificity is observed for apoptosome formation and caspase activation, the cleavage occurring only in the presence of dATP or ATP. In this study, we demonstrate the connection between peroxidase activity and oleic acid-induced conformational transitions of cyt c and show how ATP is capable of modulating such interplay. By NMR measurement, we have demonstrated that ATP interacts with a site (S1) formed by K88, R91, and E62 and such interaction was weakened by mutation of E62, suggesting the selective role in the interaction played by the base moiety. Interestingly, the interactions of ATP and GTP with cyt c are significantly different at low nucleotide concentrations, with GTP being less effective in perturbing the S1 site and in eliciting apoptotic activity. To gain insights into the structural features of cyt c required for its pro-apoptotic activity and to demonstrate a regulatory role for ATP (compared to the effect of GTP), we have performed experiments on cell lysates by using cyt c proteins mutated on amino acid residues that, as suggested by NMR measurements, belong to S1. Thus, we provide evidence that ATP acts as an allosteric effector, regulating structural transitions among different conformations and different oxidation states of cyt c, which are endowed with apoptotic activity or not. On this basis, we suggest a previously unrecognized role for ATP binding to cyt c at low millimolar concentrations in the cytosol, beyond the known regulatory role during the oxidative phosphorylation in mitochondria.

Role of Lysines in Cytochrome c−Cardiolipin Interaction

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

Conversion of cytochrome c into a peroxidase: inhibitory mechanisms and implication for neurodegenerative diseases.

A further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H2O2. To investigate the peroxidase activity of non-native cyt c, different forms of the protein were analyzed with the aim to correlate their structural features with the acquired enzymatic activity and apoptogenic properties (wt cyt c/CL complex and two single cyt c variants, H26Y and Y67H, free and bound to CL). The results suggest that cyt c may respond to different environments by changing its fold thus favouring the exertion of different biological functions in different pathophysiological cell conditions. Transitions among different conformations are regulated by endogenous molecules such as ATP and may be affected by synthetic molecules such as minocycline, thus suggesting a mechanism explaining its use as therapeutic agent impacting on disease-associated oxidative and apoptotic mechanisms.

The effects of ATP and sodium chloride on the cytochrome c–cardiolipin interaction: The contrasting behavior of the horse heart and yeast proteins

In cells a portion of cytochrome c (cyt c) (15-20%) is tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane. The CL-bound protein, which has nonnative tertiary structure, altered heme pocket, and disrupted Fe(III)-M80 axial bond, is thought to play a role in the apoptotic process. This has attracted considerable interest in order to clarify the mechanisms governing the cyt c-CL interaction. Herein we have investigated the binding reaction of CL with the c-type cytochromes from horse heart and yeast. Although the two proteins possess a similar tertiary architecture, yeast cyt c displays lower stability and, contrary to the equine protein, it does not bind ATP and lacks pro-apoptotic activity. The study has been performed in the absence and in the presence of ATP and NaCl, two compounds that influence the (horse cyt c)-CL binding process and, thus, the pro-apoptotic activity of the protein. The two proteins behave differently: while CL interaction with horse cyt c is strongly influenced by the two effectors, no effect is observed for yeast cyt c. It is noteworthy that NaCl induces dissociation of the (horse cyt c)-CL complex but has no influence on that of yeast cyt c. The differences found for the two proteins highlight that specific structural factors, such as the different local structure conformation of the regions involved in the interactions with either CL or ATP, can significantly affect the behavior of cyt c in its reaction with liposomes and the subsequent pro-apoptotic action of the protein.

ΔNp63 targets cytoglobin to inhibit oxidative stress-induced apoptosis in keratinocytes and lung cancer

During physiological aerobic metabolism, the epidermis undergoes significant oxidative stress as a result of the production of reactive oxygen species (ROS). To maintain a balanced oxidative state, cells have developed protective antioxidant systems, and preliminary studies suggest that the transcriptional factor p63 is involved in cellular oxidative defence. Supporting this hypothesis, the ΔNp63α isoform of p63 is expressed at high levels in the proliferative basal layer of the epidermis. Here we identify the CYGB gene as a novel transcriptional target of ΔNp63 that is involved in maintaining epidermal oxidative defence. The CYGB gene encodes cytoglobin, a member of the globin protein family, which facilitates the diffusion of oxygen through tissues and acts as a scavenger for nitric oxide or other ROS. By performing promoter activity assays and chromatin immunoprecipitation, reverse transcriptase quantitative PCR and western blotting analyses, we confirm the direct regulation of CYGB by ΔNp63α. We also demonstrate that CYGB has a protective role in proliferating keratinocytes grown under normal conditions, as well as in cells treated with exogenous hydrogen peroxide. These results indicate that ΔNp63, through its target CYGB has an important role in the cellular antioxidant system and protects keratinocytes from oxidative stress-induced apoptosis. The ΔNp63–CYGB axis is also present in lung and breast cancer cell lines, indicating that CYGB-mediated ROS-scavenging activity may also have a role in epithelial tumours. In human lung cancer data sets, the p63–CYGB interaction significantly predicts reduction of patient survival.

Cooperative homodimeric hemoglobin from Scapharca inaequivalvis. cDNA cloning and expression of the fully functional protein in E. coli.

The overexpression of the fully functional, cooperative homodimeric hemoglobin of the bivalve mollusc. Scapharca inaequivalvis, has been accomplished in E. coli from its cDNA. The latter was isolated by PCR amplification of total RNA and sequenced. The cDNA‐derived sequence differed by a single amino acid when compared to that previously obtained from purified protein. Interest in this hemoglobin resides in the unique assemblage of the two identical subunits, with the heme groups facing each other in the inside of the molecule, opposite to that occurring in vertebrate hemoglobins. The results presented here are the basis for future studies of structure/function relationships by site directed mutagenesis.

Setdb1, a novel interactor of ΔNp63, is involved in breast tumorigenesis

ΔNp63 has been recently involved in self-renewal potential of breast cancer stem cells. Although the p63 transcriptional profile has been extensively characterized, our knowledge of the p63-binding partners potentially involved in the regulation of breast tumour progression is limited. Here, we performed the yeast two hybrid approach to identify p63α interactors involved in breast tumorigenesis and we found that SETDB1, a histone lysine methyl transferases, interacts with ΔNp63α and that this interaction contributes to p63 protein stability. SETDB1 is often amplified in primary breast tumours, and its depletion confers to breast cancer cells growth disadvantage. We identified a list of thirty genes repressed by ΔNp63 in a SETDB1-dependent manner, whose expression is positively correlated to survival of breast cancer patients. These results suggest that p63 and SETDB1 expression, together with the repressed genes, may have diagnostic and prognostic potential.

Salt-induced formation of the A-state of ferricytochrome c – effect of the anion charge on protein structure

Structural information on partially folded forms is important for a deeper understanding of the folding mechanism(s) and the factors affecting protein stabilization. The non‐native compact state of equine cytochrome c stabilized by salts in an acidic environment (pH 2.0–2.2), called the A‐state, is considered a suitable model for the molten globule of cytochrome c, as it possesses a native‐like α‐helix conformation but a fluctuating tertiary structure. In this article, we extend our knowledge on anion‐induced protein stabilization by determining the effect of anions carrying a double negative charge; unlike monovalent anions (which are thought to exert an ‘ionic atmosphere’ effect on the macromolecule), divalent anions are thought to bind to the protein at specific surface sites. Our data indicate that divalent anions, in comparison to monovalent ions, have a greater tendency to stabilize the native‐like M–Fe(III)–H coordinated state of the protein. The possibility that divalent anions may bind to the protein at the same sites previously identified for polyvalent anions was evaluated. To investigate this issue, the behavior of the K88E, K88E/T89K and K13N mutants was investigated. The data obtained indicate that the mutated residues, which contribute to form the binding sites of polyanions, are important for stabilization of the native conformation; the mutants investigated, in fact, all show an increased amount of the misligated H–Fe(III)–H state and, with respect to wild‐type cytochrome c, appear to be less sensitive to the presence of the anion. These residues also modulate the conformation of unfolded cytochrome c, influencing its spin state and the coordination to the prosthetic group.