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.

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.

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.

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.

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.

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.

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.

Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection.

Background: Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms.

Differential proteomic analysis of subfractioned human hepatocellular carcinoma tissues.

To discover new potential biomarkers of HCC, we used 2-DE gel separation and MALDI-TOF-MS analysis of partially enriched nuclear fractions from liver biopsies of 20 different patients. We obtained a proteomic map of subfractioned liver samples including about 200 common protein spots, among which identified components corresponded to expression products of 52 different genes. A differential analysis of proteins from tumoral and control tissues revealed a significant change in the expression level of 16 proteins associated to cytoskeletal, stress response and metabolic functions.

Transcriptome and proteome analysis of osteocytes treated with nitrogen-containing bisphosphonates.

We combined high-throughput screening of differential mRNAs with mass spectrometric characterization of proteins obtained from osteocytes untreated and treated with Risedronate. Microarray analysis revealed, upon treatment, a marked upregulation of messengers encoding zinc-proteins. MS analysis identified 84 proteins in the osteocytes proteome map.

APE1/Ref-1 interacts with NPM1 within nucleoli and plays a role in the rRNA quality control process.

APE1/Ref-1 (hereafter, APE1), a DNA repair enzyme and a transcriptional coactivator, is a vital protein in mammals. Its role in controlling cell growth and the molecular mechanisms that fine-tune its different cellular functions are still not known. By an unbiased proteomic approach, we have identified and characterized several novel APE1 partners which, unexpectedly, include a number of proteins involved in ribosome biogenesis and RNA processing.

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.

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.

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.

The solution structure of DNA-free Pax-8 paired box domain accounts for redox regulation of transcriptional activity in the pax protein family.

Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redox-based mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys45 and Cys57). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain.

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.

Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma: possible prognostic significance.

APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection.

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.

Altered intracellular redox status in Gaucher disease fibroblasts and impairment of adaptive response against oxidative stress.

Gaucher disease (GD) is a lysosomal storage disorder, due to glucosylceramide (GlcCer) accumulation in several body tissues, which causes cellular failure by yet unidentified mechanisms. Several evidence indicates that GD pathogenesis is associated to an impairment in intracellular redox state. In fibroblast primary cultures, reactive oxygen species (ROS) levels and protein carbonyl content resulted significantly increased in GD patients compared to healthy donors, suggesting that GD cells, facing a condition of chronic oxidative stress, have evolved an adaptive response to survive.

Both vitamin B6 and total homocysteine plasma levels predict long-term atherothrombotic events in healthy subjects.

Aims: The contribution of homocysteine and group B vitamins in determining cardiovascular risk is debated. We assessed the predictive value of total homocysteine (tHcy), vitamin B12, folate, and vitamin B6 on the long-term occurrence of coronary and cerebral atherothrombotic events in a nested case-control study.

Methods And Results: Within a cohort of 1021 healthy subjects (490 men and 531 women) recruited in 1987, 66 first-ever coronary and 43 first-ever cerebrovascular events were recorded at a 12-year follow-up (cases, n=109).

Oxidized transthyretin in amniotic fluid as an early marker of preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive syndrome and a major cause of maternal and fetal morbidity and mortality. At the present time, no reliable screening tests to identify women at risk are available. We have compared the amniotic fluids (AF) proteomic maps of five preeclamptic patients with those of five controls.

Nucleotide receptors stimulation by extracellular ATP controls Hsp90 expression through APE1/Ref-1 in thyroid cancer cells: a novel tumorigenic pathway.

Nucleotide receptors signaling affects cell proliferation, with possible implications on tumorigenic processes. However, molecular targets and action mechanisms of the extracellular nucleotides are still poorly elucidated. We have previously shown in ARO cells that APE1/Ref-1, a transcriptional coactivator responsible for the maintenance of the cellular proliferative rate, is functionally controlled by P2-mediated signaling.

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.

Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation.

Knowledge of early molecular events occurring upon ischemia/reperfusion (I/R) during liver transplantation (LT) is of great importance to improve the therapeutic intervention of surgical treatment. However, nowadays, few data are available on early protein targets of I/R injury. To identify these proteins, we used a differential proteomics approach in the characterization human liver biopsies during I/R upon LT.

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.