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.

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.

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.

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.

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.

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.

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.

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. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time. RESULTS: With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle. CONCLUSIONS: The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.

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. These data may provide novel candidate biomarkers for HCC and useful insights for understanding the mechanisms of HCC pathogenesis and progression.

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. In particular, a novel interaction between nucleophosmin (NPM1) and APE1 was characterized. We observed that the 33 N-terminal residues of APE1 are required for stable interaction with the NPM1 oligomerization domain. As a consequence of the interaction with NPM1 and RNA, APE1 is localized within the nucleolus and this localization depends on cell cycle and active rRNA transcription. NPM1 stimulates APE1 endonuclease activity on abasic double-stranded DNA (dsDNA) but decreases APE1 endonuclease activity on abasic single-stranded RNA (ssRNA) by masking the N-terminal region of APE1 required for stable RNA binding. In APE1-knocked-down cells, pre-rRNA synthesis and rRNA processing were not affected but inability to remove 8-hydroxyguanine-containing rRNA upon oxidative stress, impaired translation, lower intracellular protein content, and decreased cell growth rate were found. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through posttranscriptional mechanisms.

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. Risedronate affected the expression of 10 proteins, associated with cytoskeleton, stress-response and metabolism. Data validated using gel imaging in combination with the GLaD post digestion isotopic labeling method provide the molecular basis for understanding the role of bisphosphonates as antiapoptotic drugs for osteocytes.

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.

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.

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. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys45 and Cys57 and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors.

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.

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. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surrounding liver cirrhosis (SLC) and compared with normal liver tissue. Survival probability was evaluated using Kaplan-Meier curves (log-rank test) and Cox regression. Cytoplasmic expression of APE1/Ref-1 was significantly higher in HCC than in SLC (P = 0.00001); normal liver showed only nuclear reactivity. Patients with poorly differentiated HCC showed a cytoplasmic expression three times higher than those with well-differentiated HCC (P = 0.03). Cytoplasmic localization was associated with a median survival time shorter than those with negative cytoplasmic reactivity (0.44 compared with 1.64 years, P = 0.003), and multivariable analysis confirmed that cytoplasmic APE1/Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE1/Ref-1 is increased in HCC and is associated with a lower degree of differentiation and a shorter survival time, pointing to the use of the cytoplasmic localization of APE1/Ref-1 as a prognostic marker for HCC.

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.

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. The ROS rise is probably due to NAD(P)H oxidase activity, being inhibited by the treatment with diphenylene iodonium chloride. Interestingly, GD cells are more sensitive to H(2)O(2) induced cell death, suggesting a dysregulation in the adaptive response to oxidative stress in which APE1/Ref-1 plays a central role. We found that the cytoplasmic amounts of APE1/Ref-1 protein were significantly higher in GD fibroblasts with respect to controls, and that GD cells failed to upregulate its expression upon H(2)O(2) treatment. Both ROS and APE1/Ref-1 increases are due to GlcCer accumulation, being prevented by treatment of GD fibroblasts with Cerezyme and induced in healthy fibroblasts treated with conduritol-beta-epoxide. These data, suggesting that GD cells display an impairment in the cellular redox state and in the adaptive cellular response to oxidative stress, may open new perspectives in the comprehension of GD pathogenesis.

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). A total of 109 control subjects (remaining free from events) were matched with cases according to age, sex, smoking, hypertension, dyslipidaemia, and body mass index. Serum samples obtained in 1987 at baseline were used to measure tHcy, folate, and vitamins B12 and B6, as well as C-reactive protein plasma concentrations. We found a significant graded association between tHcy levels and the risk of coronary and cerebrovascular events [odds ratio (OR) for uppermost vs. lowermost quartile=1.34, 95% CI 1.01-1.76)]. Folate and vitamin B12 did not significantly differ between cases and controls, but were negatively (P<0.01) correlated with tHcy. Vitamin B6 did not correlate with tHcy levels, but differed significantly between cases and controls: for subjects in the uppermost quartile vs. the lowermost quartile of vitamin B6, OR=0.69 (95% CI 0.49-0.98). For subjects in the lowermost quartile of vitamin B6 and the uppermost quartile of tHcy, OR=17.50 (95% CI 1.97, 155.59). Cases and controls were not different as to C-reactive protein. CONCLUSION: tHcy and plasma vitamin B6 are long-term independent risk factors for coronary and cerebrovascular events.