Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions.

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein-protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

Understanding different functions of mammalian AP endonuclease (APE1) as a promising tool for cancer treatment.

The apurinic endonuclease 1/redox factor-1 (APE1) has a crucial function in DNA repair and in redox signaling in mammals, and recent studies identify it as an excellent target for sensitizing tumor cells to chemotherapy. APE1 is an essential enzyme in the base excision repair pathway of DNA lesions caused by oxidation and alkylation. As importantly, APE1 also functions as a redox agent maintaining transcription factors involved in cancer promotion and progression in an active reduced state. Very recently, a new unsuspected function of APE1 in RNA metabolism was discovered, opening new perspectives for this multifunctional protein. These observations underline the necessity to understand the molecular mechanisms responsible for fine-tuning its different biological functions. This survey intends to give an overview of the multifunctional roles of APE1 and their regulation in the context of considering this protein a promising tool for anticancer therapy.

A proteomic approach to the bilirubin-induced toxicity in neuronal cells reveals a protective function of DJ-1 protein.

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long-term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB-induced neuronal toxicity, we used the human neuroblastoma cell line SH-SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin-induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.

Genome-wide analysis and proteomic studies reveal APE1/Ref-1 multifunctional role in mammalian cells.

Apurinic apyrimidinic endonuclease/redox effector factor 1 (APE1/Ref-1) protects cells from oxidative stress by acting as a central enzyme in base excision repair pathways of DNA lesions and through its independent activity as a redox transcriptional co-activator. Dysregulation of this protein has been associated with cancer development. At present, contrasting data have been published regarding the biological relevance of the two functions as well as the molecular mechanisms involved. Here, we combined both mRNA expression profiling and proteomic analysis to determine the molecular changes associated with APE1 loss-of-expression induced by siRNA technology. This approach identified a role of APE1 in cell growth, apoptosis, intracellular redox state, mitochondrial function, and cytoskeletal structure. Overall, our data show that APE1 acts as a hub in coordinating different and vital functions in mammalian cells, highlighting the molecular determinants of the multifunctional nature of APE1 protein.

APE1/Ref-1 regulates PTEN expression mediated by Egr-1.

APE1/Ref-1, the mammalian ortholog of E. coli Xth, and a multifunctional protein possessing both DNA repair and transcriptional regulatory activities, has dual role in controlling cellular response to oxidative stress. It is rate-limiting in repair of oxidative DNA damage including strand breaks and also has co-transcriptional activity by modulating genes expression directly regulated by Egr-1 and p53 transcription factors. PTEN, a phosphoinositide phosphatase, acts as an 'off' switch in the PI-3 kinase/Akt signalling pathway and regulates cell growth and survival. It is shown here that transient alteration in the APE1 level in HeLa cells modulates PTEN expression and that acetylatable APE1 is required for the activation of the PTEN gene. Acetylation of APE1 enhances its binding to distinct trans-acting complexes involved in activation or repression. The acetylated protein is deacetylated in vivo by histone deacetylases. It was found that exposure of HeLa cells to H(2)O(2) and to histone deacetylase inhibitors increases acetylation of APE1 and induction of PTEN. The absence of such induction in APE1-downregulated HeLa cells confirmed APE1's role in regulating inducible PTEN expression. That APE1-dependent PTEN expression is mediated by Egr-1 was supported by experiments with cells ectopically expressing Egr-1. Thus, the data open new perspectives in the comprehension of the many functions exerted by APE1 in controlling cell response to oxidative stress.

Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway.

Unconjugated bilirubin (UCB) is the major degradation product of the heme catabolism. A growing body of evidences suggests that UCB plays major biological effects by inhibiting cell proliferation in cancer cell lines and eliciting cell toxicity particularly in neurons and glial cells. Early molecular events responsible for bilirubin-induced cytotoxicity remain poorly understood. Using HeLa cells and mouse embryonic fibroblasts, we found that UCB at a concentration of free pigment (Bf) of 80 nM induced oxidative stress, promoting a significant increase in intracellular reactive oxygen species (ROS) and a decreased cell survival (by the MTT test). The ROS increase activated the antioxidant cell response through APE1/Ref-1, a master redox regulator in eukaryotic cells. Activation of APE1/Ref-1 was followed by a concomitant activation of Egr-1 transcription factor and by an upregulation of PTEN tumor suppressor, an Egr-1 target gene, leading to inhibition of cell growth. Blocking ROS generation with N-acetylcysteine pretreatment, restored cell survival, limited the upregulation of PTEN in response to UCB, and prevented the inhibition of cell proliferation. HeLa cells transfected with mutants of the PTEN promoter or silenced with APE1/Ref-1 small interference RNA confirmed that UCB modulates a signaling pathway involving APE1/Ref-1, Egr-1, and PTEN. These findings describe a new molecular pathway involved in the cytotoxic effects of UCB.

Activation of APE1/Ref-1 is dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP.

Apurinic apyrimidinic endonuclease redox effector factor-1 (APE1/Ref-1) is involved both in the base excision repair (BER) of DNA lesions and in the eukaryotic transcriptional regulation. APE1/Ref-1 is regulated at both the transcriptional and post-translational levels, through control of subcellular localization and post-translational modification. In response to stress conditions, several cell types release ATP, which exerts stimulatory effects on eukaryotic cells via the purinergic receptors (P2) family. By using western blot and immunofluorescence analysis on a human tumour thyroid cell line (ARO), we demonstrate that purinergic stimulation by extracellular ATP induces quick cytoplasm to nucleus translocation of the protein at early times and its neosynthesis at later times. Continuous purinergic triggering by extracellular ATP released by ARO cells is responsible for the control of APE1/Ref-1 intracellular level. Interference with intracellular pathways activated by P2 triggering demonstrates that Ca2+ mobilization and intracellular reactive oxygen species (ROS) production are responsible for APE1/Ref-1 translocation. The APE1/Ref-1 activities on activator protein-1 (AP-1) DNA binding and DNA repair perfectly match its nuclear enrichment upon ATP stimulation. The biological relevance of our data is reinforced by the observation that APE1/Ref-1 stimulation by ATP protects ARO cells by H2O2-induced cell death. Our data provide new insights into the complex mechanisms regulating APE1/Ref-1 functions.

Bisphosphonates activate nucleotide receptors signaling and induce the expression of Hsp90 in osteoblast-like cell lines.

Bisphosphonates are the most important drugs used in the treatment of osteoporosis as they inhibit osteoclast resorption and stimulate proliferation of osteoblasts. However, the molecular mechanisms responsible for these effects are still poorly elucidated. It is known that nucleotide receptors-mediated signaling plays a central role in modulating osteoblasts growth in response to mechanical stress. By using osteoblast-like cell lines (i.e., HOBIT, MG-63, ROS P2Y), which express P2Y receptors, we found that the treatment with risedronate promotes non-lytic ATP release leading to activation of ERKs through the involvement of P2Y receptors triggering. A major role in this signal transduction pathway seems to be the involvement of P2Y(1) and P2Y(2) receptors, since the stimulatory effect of risedronate on ERKs is not appreciable in ROS 17/2.8 cells, which do not express these two receptors. Differential proteomics analysis identified Hsp90 upregulation as a result of risedronate effect on HOBIT and MG-63 cells. The stimulatory effect is dependent on ERKs activation involving nucleotide receptors triggering and leads to increased proliferation of osteoblast-like cells. In fact, functional inactivation of Hsp90 by the specific inhibitor 17-AAG prevents the bisphosphonate-induced mitogenic effects in osteoblasts. These findings show that bisphosphonates, by inducing ATP release, may also act through nucleotide receptors signaling leading to ERKs activation and may exert their mitogenic role on osteoblasts through the involvement of Hsp90.

Differential proteomic analysis of nuclear extracts from thyroid cell lines.

Nuclear proteins play a major role in controlling cell functions. Differential proteomic analysis of nuclear proteins by combined 2D gel electrophoresis (2D-E) and mass spectrometry procedures can provide useful information to understand the control of cell proliferation and differentiation. To identify proteins involved in dedifferentiation, we used a differential proteomics approach by comparing nuclear extracts from the differentiated rat thyroid cell line FRTL-5 and the derived undifferentiated Ki-mol cell line, obtained by transformation with the Ki-ras oncogene. Thirteen proteins were identified as differently expressed in the nuclear compartment between the two cell lines. RT-PCR analysis performed on seven differently expressed genes showed that only in two cases the difference may be ascribable to a transcriptional mechanism. Since one of the identified proteins, namely apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1), is suspected to play a role in thyroid tumorigenesis, we used a glutathione S-transferase (GST)-pulldown assay coupled to a 2D electrophoretic/matrix assisted laser desorption ionization-time of flight (MALDI-TOF)-mass spectrometry (MS) analysis to detect and identify its interacting partners. We show here that beta-actin directly interacted with APE1/Ref-1, as confirmed by co-immunoprecipitation assays and that this interaction was enhanced by oxidative stress on FRTL-5 cells.

Antiproliferative effect in chronic myeloid leukaemia cells by antisense peptide nucleic acids.

Peptide nucleic acid (PNA) is a synthetic DNA analogue that is resistant to nucleases and proteases and binds with exceptional affinity to RNA. Because of these properties PNA has the potential to become a powerful therapeutic agent to be used in vivo. Until now, however, the use of PNA in vivo has not been much investigated. Here, we have attempted to reduce the expression of the bcr/abl oncogene in chronic myeloid leukaemia KYO-1 cells using a 13mer PNA sequence (asPNA) designed to hybridise to the b2a2 junction of bcr/abl mRNA. To enhance cellular uptake asPNA was covalently linked to the basic peptide VKRKKKP (NLS-asPNA). Moreover, to investigate the cellular uptake by confocal microscopy, both PNAs were linked by their N-terminus to fluorescein (FL). Studies of uptake, carried out at 4 and 37 degrees C on living KYO-1 cells stained with hexidium iodide, showed that both NLS-asPNA-FL and asPNA-FL were taken up by the cells, through a receptor-independent mechanism. The intracellular amount of NLS-asPNA-FL was about two to three times higher than that of asPNA-FL. Using a semi-quantitative RT- PCR technique we found that 10 micro M asPNA and NLS-asPNA reduced the level of b2a2 mRNA in KYO-1 cells to 20 +/- 5% and 60 +/- 10% of the control, respectively. Western blot analysis showed that asPNA promoted a significant inhibition of p210(BCR/ABL) protein: residual protein measured in cells exposed for 48 h to asPNA was approximately 35% of the control. Additionally, asPNA impaired cell growth to 50 +/- 5% of the control and inhibited completion of the cell cycle. In summary, these results demonstrate that a PNA 13mer is taken up by KYO-1 cells and is capable of producing a significant and specific down-regulation of the bcr/abl oncogene involved in leukaemogenesis.

Extracellular nucleotides activate Runx2 in the osteoblast-like HOBIT cell line: a possible molecular link between mechanical stress and osteoblasts' response.

Dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production, by acting at the gene expression level. Molecular mechanisms through which mechanical forces are conversed into biochemical signalling in bone are still poorly understood. A growing body of evidence point to extracellular nucleotides (i.e., ATP and UTP) as soluble factors released in response to mechanical stimulation in different cell systems. Runx2, a fundamental transcription factor involved in controlling osteoblasts differentiation, has been recently identified as a target of mechanical signals in osteoblastic cells. We tested the hypothesis that these extracellular nucleotides could be able to activate Runx2 in the human osteoblastic HOBIT cell line. We found that ATP and UTP treatments, as well as hypotonic stress, promote a significant stimulation of Runx2 DNA-binding activity via a mechanism involving PKC and distinct mitogen-activated protein kinase cascades. In fact, by using the specific inhibitors SB203580 (specific for p38 MAPK) and PD98059 (specific for ERK-1/2 MAPK), we found that ERK-1/2, but not p38, play a major role in Runx2 activation. On the contrary, another important transcription factor, i.e., Egr-1, that we previously demonstrated being activated by extracellular released nucleotides in this osteoblastic cell line, demonstrated to be susceptible to both ERK-1/2 and p38 kinases. These data suggest a possible differential involvement of these two transcription factors in response to extracellularly released nucleotides. The biological relevance of our data is strengthened by the finding that a target gene of Runx2, i.e., Galectin-3, is up-regulated by ATP stimulation of HOBIT cells with a comparable kinetic of that found for Runx2. Since it is known that osteocytes are the primary mechanosensory cells of the bone, we hypothesize that they may signal mechanical loading to osteoblasts through release of extracellular nucleotides. Altogether, these data suggest a molecular mechanism explaining the purinoreceptors-mediated activation of specific gene expression in osteoblasts and could be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Anti-gene peptide nucleic acid targeted to proviral HIV-1 DNA inhibits in vitro HIV-1 replication.

Highly active antiretroviral therapy (HAART) is unlikely to affect reservoirs of HIV in latently infected cells. Anti-gene compounds, such as peptide nucleic acids (PNAs), which block transcriptional activity via sequence-specific invasion of double-stranded DNA may be an effective strategy to target cells harbouring proviral HIV DNA. Here we show that a PNA oligomer (PNA(HIV)), 15 bases in length, linked to a nuclear localization signal (NLS), substantially suppressed HIV-1 replication in chronically infected lymphocytes and macrophages and efficiently prevented mitogen-induced HIV-1 reactivation in lymphocytes, as determined by HIV-p24 antigen production in supernatants and FACS analysis for intracellular HIV accumulation. In contrast, a mismatched PNA did not show any effect on HIV expression. Semi-quantitative RT-PCR and quantitative real-time RT-PCR demonstrated a decrease of HIV RNA expression in infected cells treated by PNA(HIV) indicating that inhibition of HIV-1 replication occurred at the transcription step. In conclusion, the use of anti-gene PNA to target the HIV-1 proviral DNA in the quest for new antiretroviral agents appears quite promising.

Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver.

Oxidative stress is a major pathogenetic event occurring in several liver disorders and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. While several markers of chronic oxidative stress are well known, early protein targets of oxidative injury are not well defined. In order to identify these proteins, we used a differential proteomics approach to HepG2 human liver cells treated for 10 min with 500 microM H(2)O(2). This dose was sufficient to induce a slight decrease of total GSH and total protein thiol content without affecting cell viability. By performing Differential Proteomic analysis, by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins which resulted sensitive to H(2)O(2) treatment. The main changes were due to post-translational modifications of native polypeptides. Three of these proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, namely PrxI, PrxII and PrxVI, that showed changes in their pI as result of overoxidation. Mass mapping experiments demonstrated the specific modification of peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining the increase in negative charge measured for these proteins. The oxidation kinetic of all peroxiredoxins was extremely rapid and sensitive, occurring at H(2)O(2) doses unable to affect the common markers of cellular oxidative stress. Recovery experiments demonstrated a quite different behaviour between 1-Cys and 2-Cys containing Prxs as their retroreduction features is concerned, thus suggesting a functional difference between different class of Prxs. The in vivo relevance of our study is demonstrated by the finding that overoxidation of PrxI occurs during I/R upon liver transplantation and is dependent on the time of warm ischemia. Our present data could be of relevance in setting up more standardized procedures to preserve organs for transplantations.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: evidence for an autoregulatory loop.

The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H(2)O(2) stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H(2)O(2) stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells.

Apurinic/apyrimidinic endonuclease 1 (APE1) is the main abasic endonuclease in the base excision repair (BER) pathway of DNA lesions caused by oxidation/alkylation in mammalian cells; within nucleoli it interacts with nucleophosmin and rRNA through N-terminal Lys residues, some of which (K(27)/K(31)/K(32)/K(35)) may undergo acetylation in vivo. Here we study the functional role of these modifications during genotoxic damage and their in vivo relevance. We demonstrate that cells expressing a specific K-to-A multiple mutant are APE1 nucleolar deficient and are more resistant to genotoxic treatment than those expressing the wild type, although they show impaired proliferation. Of interest, we find that genotoxic treatment induces acetylation at these K residues. We also find that the charged status of K(27)/K(31)/K(32)/K(35) modulates acetylation at K(6)/K(7) residues that are known to be involved in the coordination of BER activity through a mechanism regulated by the sirtuin 1 deacetylase. Of note, structural studies show that acetylation at K(27)/K(31)/K(32)/K(35) may account for local conformational changes on APE1 protein structure. These results highlight the emerging role of acetylation of critical Lys residues in regulating APE1 functions. They also suggest the existence of cross-talk between different Lys residues of APE1 occurring upon genotoxic damage, which may modulate APE1 subnuclear distribution and enzymatic activity in vivo.

Shotgun proteomics analysis reveals new unsuspected molecular effectors of nitrogen-containing bisphosphonates in osteocytes.

Nitrogen-containing bisphosphonates (N-BPs) are therapeutic agents used to treat osteoporosis and promote osteoblast and osteocyte survival. The molecular mechanisms underlying this effect have been extensively studied, but the global changes induced by N-BPs at the protein level are not known. In this context, we investigated the effect of 10(-7)M Risedronate for 1h and 48h on MLO-Y4 osteocytic cells, through a quantitative, label free shotgun proteomic analysis. We described herein a preliminary proteome map of untreated MLO-Y4 cells, composed of 353 protein species. Moreover, we identified 10 and 15 differentially expressed proteins after 1h and 48h of Risedronate treatment, respectively. Among these, PARK7/DJ-1 protein levels were induced up to 3 times and this event was associated with the activation of the pro-survival Akt pathway that we propose as a novel player in the effect of N-BPs on osteocytes. Risedronate was also able to induce the expression and the secretion of the growth factor pro-granulin. In addition, protein prenylation inhibition appeared to be involved in the modulation of MLO-Y4 proteome by RIS in a protein-specific manner. In conclusion, these findings unveil novel functions targeted by N-BPs in osteocytes and could be useful to design novel pharmaceutical compounds.

Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) protects cells from oxidative stress via the base excision repair pathway and as a redox transcriptional coactivator. It is required for tumor progression/metastasis, and its up-regulation is associated with cancer resistance. Loss of APE1 expression causes cell growth arrest, mitochondrial impairment, apoptosis, and alterations of the intracellular redox state and cytoskeletal structure. A detailed knowledge of the molecular mechanisms regulating its different activities is required to understand the APE1 function associated with cancer development and for targeting this protein in cancer therapy. To dissect these activities, we performed reconstitution experiments by using wild-type and various APE1 mutants. Our results suggest that the redox function is responsible for cell proliferation through the involvement of Cys-65 in mediating APE1 localization within mitochondria. C65S behaves as a loss-of-function mutation by affecting the in vivo folding of the protein and by causing a reduced accumulation in the intermembrane space of mitochondria, where the import protein Mia40 specifically interacts with APE1. Treatment of cells with (E)-3-(2-[5,6-dimethoxy-3-methyl-1,4-benzoquinonyl])-2-nonyl propenoic acid, a specific inhibitor of APE1 redox function through increased Cys-65 oxidation, confirm that Cys-65 controls APE1 subcellular trafficking and provides the basis for a new role for this residue.

The many functions of APE1/Ref-1: not only a DNA repair enzyme.

APE1/Ref-1 (APE1), the mammalian ortholog of Escherichia coli Xth, and a multifunctional protein possessing both DNA repair and transcriptional regulatory activities, has a pleiotropic role in controlling cellular response to oxidative stress. APE1 is the main apurinic/apyrimidinic endonuclease in eukaryotic cells, playing a central role in the DNA base excision repair pathway of all DNA lesions (uracil, alkylated and oxidized, and abasic sites), including single-strand breaks, and has also cotranscriptional activity by modulating genes expression directly regulated by either ubiquitous (i.e., AP-1, Egr-1, NFkappa-B, p53, and HIF) and tissue specific (i.e., PEBP-2, Pax-5 and -8, and TTF-1) transcription factors. In addition, it controls the intracellular redox state by inhibiting the reactive oxygen species (ROS) production. At present, information is still inadequate regarding the molecular mechanisms responsible for the coordinated control of its several activities. Both expression and/or subcellular localization are altered in several metabolic and proliferative disorders such as in tumors and aging. Here, we have attempted to coalesce the most relevant information concerning APE1's different functions in order to shed new light and to focus current and future studies to fully understand this unique molecule that is acquiring more and more interest and translational relevance in the field of molecular medicine.

Modern strategies to identify new molecular targets for the treatment of liver diseases: The promising role of Proteomics and Redox Proteomics investigations.

Oxidative stress, due to an imbalance between the generation of ROS and the antioxidant defense capacity of the cell, is a major pathogenetic event occurring in several liver diseases, ranging from metabolic to proliferative. Main sources of ROS are represented by mitochondria and cytochrome P450 enzymes in the hepatocytes, Küppfer cells, and neutrophils. Oxidative stress affects major cellular components including lipids, DNA, and proteins. Through modulation of protein structure/function, ROS can influence gene expression profile by affecting intracellular signal transduction pathways. While several enzymatic and nonenzymatic markers of chronic oxidative stress are well known in liver, early protein targets of oxidative injury are yet poorly defined. Identification of these biomarkers will enable early detection of liver diseases and will allow monitoring the degree of liver damage, the response to pharmacological therapies, and the development of new therapeutic approaches. In the era of molecular medicine, new proteomic methodologies promise to establish a relationship between pathological hallmarks of the disease and protein structural/functional modifications, thus allowing a better understanding and a more rational therapy on liver disorders. Purpose of this review is to critically analyze the application of proteomic and redox proteomic approaches to the study of oxidative stress-linked liver diseases.

APE1/Ref-1 in Alzheimer's disease: an immunohistochemical study.

The oxidative injury in Alzheimer's disease (AD), in which amyloid beta protein induces production of reactive oxygen species, may be cause of neurodegeneration. APE1/Ref-1 is a protein involved in DNA repair and in redox co-activating function over different transcription factors. We investigated by immunohistochemistry using a highly specific monoclonal antibody, the localization of APE1/Ref-1 in autoptic and bioptic AD brain tissues in comparison with brains with unrelated pathological or normal conditions. Reliable APE1/Ref-1 immunostaining was obtained in biopsies, but not in autoptic tissues. An increased nuclear expression of APE1/Ref-1 in AD cerebral cortex supports the view that the cellular adaptive response to the oxidative stress condition is involved in the pathogenesis of this disease.