One- and Two-Electron Oxidations of β-Amyloid_25-35 by Carbonate Radical Anion (CO₃•-) and Peroxymonocarbonate (HCO₄-):Role of Sulfur in Radical Reactions and Peptide Aggregation

The β-amyloid (Aβ) peptide plays a key role in the pathogenesis of Alzheimer’s disease. The methionine (Met) residue at position 35 in Aβ C-terminal domain is critical for neurotoxicity, aggregation, and free radical formation initiated by the peptide. The role of Met in modulating toxicological properties of Aβ most likely involves an oxidative event at the sulfur atom. We therefore investigated the one- or two-electron oxidation of the Met residue of Aβ25-35 fragment and the effect of such oxidation on the behavior of the peptide. Bicarbonate promotes two-electron oxidations mediated by hydrogen peroxide after generation of peroxymonocarbonate (HCO4−, PMC). The bicarbonate/carbon dioxide pair stimulates one-electron oxidations mediated by carbonate radical anion (CO3•−). PMC efficiently oxidizes thioether sulfur of the Met residue to sulfoxide. Interestingly, such oxidation hampers the tendency of Aβ to aggregate. Conversely, CO3•− causes the one-electron oxidation of methionine residue to sulfur radical cation (MetS•+). The formation of this transient reactive intermediate during Aβ oxidation may play an important role in the process underlying amyloid neurotoxicity and free radical generation

Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

<p>Abstract</p> <p>Background</p> <p>Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype.</p> <p>Methods</p> <p>The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored.</p> <p>Results</p> <p>We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.</p> <p>Conclusion</p> <p>Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.</p

Effects of UVB-induced oxidative stress on protein expression and specific protein oxidation in normal human epithelial keratinocytes: a proteomic approach

<p>Abstract</p> <p>Background</p> <p>The UVB component of solar ultraviolet irradiation is one of the major risk factors for the development of skin cancer in humans. UVB exposure elicits an increased generation of reactive oxygen species (ROS), which are responsible for oxidative damage to proteins, DNA, RNA and lipids. In order to examine the biological impact of UVB irradiation on skin cells, we used a parallel proteomics approach to analyze the protein expression profile and to identify oxidatively modified proteins in normal human epithelial keratinocytes.</p> <p>Results</p> <p>The expression levels of fifteen proteins - involved in maintaining the cytoskeleton integrity, removal of damaged proteins and heat shock response - were differentially regulated in UVB-exposed cells, indicating that an appropriate response is developed in order to counteract/neutralize the toxic effects of UVB-raised ROS. On the other side, the redox proteomics approach revealed that seven proteins - involved in cellular adhesion, cell-cell interaction and protein folding - were selectively oxidized.</p> <p>Conclusions</p> <p>Despite a wide and well orchestrated cellular response, a relevant oxidation of specific proteins concomitantly occurs in UVB-irradiated human epithelial Keratinocytes. These modified (i.e. likely dysfunctional) proteins might result in cell homeostasis impairment and therefore eventually promote cellular degeneration, senescence or carcinogenesis.</p

Oxidative Stress in HPV-Driven Viral Carcinogenesis: Redox Proteomics Analysis of HPV-16 Dysplastic and Neoplastic Tissues

Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not per se sufficient to induce tumour development. Oxidative stress (OS) represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. To gain insight into the role of OS in cervical cancer, HPV-16 positive tissues were collected from patients with invasive squamous cervical carcinoma, from patients with High Grade dysplastic HPV lesions and from patients with no clinical evidence of HPV lesions. After virological characterization, modulation of proteins involved in the redox status regulation was investigated. ERp57 and GST were sharply elevated in dysplastic and neoplastic tissues. TrxR2 peaked in dysplastic samples while iNOS was progressively reduced in dysplastic and neoplastic samples. By redox proteomic approach, five proteins were found to have increased levels of carbonyls in dysplastic samples respect to controls namely: cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH. In carcinoma samples the peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, Annexin 2 and GAPDH were found less oxidized than in dysplastic tissues. HPV16 neoplastic progression seems associated with increased oxidant environment. In dysplastic tissues the oxidative modification of DNA and proteins involved in cell morphogenesis and terminal differentiation may provide the conditions for the neoplastic progression. Conversely cancer tissues seem to attain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/pro-survival proteins

Oxidative Stress in HPV-Driven Viral Carcinogenesis: Redox Proteomics Analysis of HPV-16 Dysplastic and Neoplastic Tissues

Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not per se sufficient to induce tumour development. Oxidative stress (OS) represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. To gain insight into the role of OS in cervical cancer, HPV-16 positive tissues were collected from patients with invasive squamous cervical carcinoma, from patients with High Grade dysplastic HPV lesions and from patients with no clinical evidence of HPV lesions. After virological characterization, modulation of proteins involved in the redox status regulation was investigated. ERp57 and GST were sharply elevated in dysplastic and neoplastic tissues. TrxR2 peaked in dysplastic samples while iNOS was progressively reduced in dysplastic and neoplastic samples. By redox proteomic approach, five proteins were found to have increased levels of carbonyls in dysplastic samples respect to controls namely: cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH. In carcinoma samples the peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, Annexin 2 and GAPDH were found less oxidized than in dysplastic tissues. HPV16 neoplastic progression seems associated with increased oxidant environment. In dysplastic tissues the oxidative modification of DNA and proteins involved in cell morphogenesis and terminal differentiation may provide the conditions for the neoplastic progression. Conversely cancer tissues seem to attain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/pro-survival proteins

β-AMYLOID25-35 MODIFICATION BY OXIDANTS DERIVED FROM BICARBONATE/CARBON DIOXIDE PAIR

The β-amyloid plays a key role in the pathogenesis of Alzheimer’s disease and contains a methionine residue at position 35 in its C-terminal domain which is critical for neurotoxicity, aggregation and free radical formation initiated by the peptide. The role of methionine in the toxicological properties of β-amyloid most likely involves an oxidative event at the sulfur atom. Many recent studies have demonstrated that the biologically ubiquitous bicarbonate/carbon dioxide pair stimulates oxidation, peroxidation and nitration of several biological targets. Hence, it is appropriate and timely to recognize that the main physiological buffer is active in redox processes. At physiological level, the bicarbonate/carbon dioxide pair, stimulates one-electron oxidations mediated by peroxynitrite and SOD with the intermediacy of carbonate anion radical (CO3•−). Furthermore, bicarbonate promotes two-electron oxidations mediated by hydrogen peroxide after generation of peroxymonocarbonate (HCO4−). We therefore investigated the ability of peroxymonocarbonate and carbonate anion radical to oxidatively modify methionine residue of β-amyloid and the effect of such oxidations on the behaviour of the β-amyloid25‒35 fragment. Peroxymonocarbonate efficiently oxidizes thioether sulfur of methionine (Met) residue of β-amyloid to sulfoxide (MetSO). Interestingly, such oxidation counteracts the tendency of β-amyloid to aggregate. Conversely, carbonate anion radical causes the one-electron oxidation of methionine residue to its radical cation (MetS•+). The formation of this transient reactive intermediate during β-amyloid25‒35 fragment oxidation may play an important role in the process underlying β-amyloid neurotoxicity and free radical generatio

Biological response of human diploid keratinocytes to quinone-producing compounds: role of NAD(P)H : quinone oxidoreductase 1

Abstract Reactive oxygen species (ROS) and quinones are known to determine redox balance alteration, oxidative stress and carcinogenicity. Keratinocytes of the human epidermis, a tissue particularly exposed to oxidant stimuli, possess a wide range of antioxidant and detoxifying mechanisms aimed to avoid oxidative damage of the tissue. In the present study, we evaluate the response of diploid and transformed human keratinocytes to exposure to L-dopa and tetrahydropapaveroline (THP), catechol compounds susceptible to undergo oxidation to form quinones with concomitant production of reactive oxygen species. We demonstrated that these compounds elicit up-regulation of intracellular antioxidant enzymes, in a different degree in normal cells with respect to transformed ones. Normal diploid keratinocytes adequately scavenge toxic substances through the activation of several, concurrent pathways. Conversely, in transformed cells, the whole oxidative burden must be detoxified by the limited set of conserved pathways that, accordingly, have to be highly activated. The biological response to catechol toxicity appears to rely on the pathway of NAD(P)H:quinone oxidoreductase 1 (NQO1). In conclusion, NAD(P)H:quinone oxidoreductase 1 confirms its antioxidant and detoxifying role contributing to the capacity of keratinocytes to protect epidermis against oxidative stress. Being retained in almost any cell, it represents a mechanism of general relevance in cell physiology

Interaction of enkephalin derivatives with reactive oxygen species

The oxidation of opioid peptides by tyrosinase in the presence of an excess of a thiol gives rise to cysteinyldopa derivatives. The major products arising from the reaction between Leu-enkephalin and cysteine are represented by 5-S-cysteinyldopaenkephalin (5-CDenk) and 2-S-cysteinyldopaenkephalin (2-CDenk). The interaction of 5-CDenk and 2-CDenk with reactive oxygen species (ROS) has been studied. These compounds are able to scavenge superoxide anion, hydroxyl and peroxyl radicals as well as to reduce the lipid peroxidation rate induced by ABAP. The scavenging activities in all instances are dose-dependent. In some cases CDenks are more active than compounds recognized as strong radical scavengers, such as Trolox and mannitol. As a result of the action of the Fenton system, the CDenks (as well as the Enks) are oxidized into pigmented derivatives. The possible implications of the interaction of CDenks and Enks with ROS on melanization process in Parkinson's disease are discussed. (C) 2001 Elsevier Science B.V. All rights reserved

Ectopic deposition of melanin pigments as detoxifying mechanism: a paradigm for basal nuclei pigmentation

Abstract Melanins are UV shielding pigments found in skin and other light exposed tissues. However, a kind of melanin, named neuromelanin (NM), is found in those deep brain loci that degenerate in Parkinson's disease (PD), where no such a function may be imagined. The NM synthetic pathway, different from the one of eumelanin based on tyrosinase, is still obscure as well as its physiological function. Here we show that under conditions of excess of toxic quinone concentration, nonmelanocytic cell strains (i.e., primary keratinocytes) may accumulate a dark cytoplasmatic pigment that proved to be a melanin. The ability of pigment deposition, possibly driven by peroxidases, is restricted to diploid cells and increases cell survival acting as a sink for potentially hazardous quinones. We suggest that in the basal nuclei, exposed to high level of catecholaminergic neurotransmitters, NM deposition is a relevant antioxidant mechanism by trapping quinones and semiquinones, thus protecting neurons from accumulating damage over many years. In this perspective, just as a hypothesis, we may imagine that PD neuron degeneration is the consequence of a reduced/abrogated ability to produce neuromelanin

Production of melanin pigments by cytochrome c/H2O2 system

In the presence of hydrogen peroxide cytochrome c can perform the oxidation of catecholamines and their S-cysteinyl-derivatives yielding melanins as final products. The initial reaction rate is linearly dependent on cytochrome c and H2O2 concentration; the reaction follows the Michaelis and Menten kinetics both for H2O2 and hydrogen donors. Sulfhydryl compounds inhibit the formation of the pigment. The reported data indicate that a heme- containing protein belonging to the mitochondrial chain can accelerate the oxidation of catecholamines to eumelanins