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Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.

It is well established that osteoblasts, the key cells involved in bone formation during development and in adult life, secrete a number of glycoproteins harboring autocrine and paracrine functions. Thus, investigating the osteoblastic secretome could yield important information for the pathophysiology of bone. In the present study, we characterized for the first time the secretome of human Hobit osteoblastic cells. We discovered that the secretome comprised 89 protein species including the powerful growth factor progranulin. Recombinant human progranulin (6 nM) induced phosphorylation of mitogen-activated protein kinase in both Habit and osteocytic cells and induced cell proliferation and survival. Notably, risedronate, a nitrogen-containing bisphosphonate widely used in the treatment of osteoporosis, induced the expression and secretion of progranulin in the Hobit secretome. In addition, our proteomic study of the Habit secretome revealed that risedronate induced the expression of ERp57, HSP60 and HSC70, three proteins already shown to be associated with the prevention of bone loss in osteoporosis. Collectively, our findings unveil novel targets of risedronate-evoked biological effects on osteoblast-like cells and further our understanding of the mechanisms of action of this currently used compound. (C) 2013 Elsevier Inc. All rights reserved.

APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-κB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-α in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-κB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-α and FAs accumulation through blockage of the redox-mediated activation of NF-κB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-α and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases.

Rs3814113 is the single-nucleotide polymorphism (SNP) showing the strongest association with high-grade serous ovarian carcinoma (HGSOC) incidence and is located in an intergenic region about 44 kb downstream of basonuclin 2 (BNC2) gene. Lifetime number of ovulations is associated with increased risk to develop HGSOC, probably because of cell damage of extrauterine Müllerian epithelium by ovulation-induced oxidative stress. However, the impact of low-penetrance HGSOC risk alleles (e.g. rs3814113) on the damage induced by oxidative stress remains unclear. Therefore, the purpose of this study was to investigate whether rs3814113 genetic interval regulates BNC2 expression and whether BNC2 expression levels impact on cell survival after oxidative stress. To do this, we analyzed gene expression levels of BNC2 first in HGSOC data sets and then in an isogenic cell line that we engineered to carry a 5 kb deletion around rs3814113. Finally, we silenced BNC2 and measured surviving cells after hydrogen peroxide (H2O2) treatment to simulate oxidative stress after ovulation. In this paper, we describe that BNC2 expression levels are reduced in HGSOC samples compared with control samples, and that BNC2 expression levels decrease following oxidative stress and ovulation in vitro and in vivo, respectively. Moreover, deletion of 5 kb surrounding rs3814113 decreases BNC2 expression levels in an isogenic cell line, and silencing of BNC2 expression levels increases cell survival after H2O2 treatment. Altogether, our findings suggest that the intergenic region located around rs3814113 regulates BNC2 expression, which in turn affects cell survival after oxidative stress response. Indeed, HGSOC samples present lower BNC2 expression levels that probably, in the initial phases of oncogenic transformation, conferred resistance to oxidative stress and ultimately reduced the clearance of cells with oxidative-induced damages.

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.

APE1/Ref-1 (also called HAP1 or APEX, and here referred to as APE1), the mammalian ortholog of Escherichia coli Xth (exonuclease III), is a vital protein (20) that acts as a master regulator of cellular response to oxidative stress conditions and contributes to the maintenance of genome stability (55, 56). APE1 is involved in both the base excision repair (BER) pathways of DNA lesions, acting as the major apurinic/apyrimidinic (AP) endonuclease, and in transcriptional regulation of gene expression as a redox coactivator of different transcription factors, such as early growth response protein 1 (Egr-1), NF-κB, and p53 (55, 56). These two biological activities are located in two functionally distinct protein domains. In fact, the N-terminal region, containing the nuclear localization signal (NLS) sequence, is principally devoted to redox activity through Cys65, while the C-terminal one exerts enzymatic activity on the abasic sites of DNA (56, 63). The protein C terminus is highly conserved during phylogenesis, while the N terminus is not. Except in mammals, which always show a high sequence conservation (more than 90%), and Danio, Drosophila, Xenopus, and Dictyostelium (presenting a sequence identity of less than 40%), the N-terminal region is mostly absent in other organisms. A third APE1 function, which is regulated by Lys6/Lys7 acetylation (7), is indirect binding to the negative calcium response elements (nCaRE) of some promoters (i.e., PTH and APE1 promoters), thus acting as a transcriptional repressor (12, 30). In different mammalian cell types, the APE1 subcellular distribution is mainly nuclear and is critical for controlling cellular proliferative rate (20, 25, 28). However, cytoplasmic, mitochondrial, and endoplasmic reticulum localization has also been reported (11, 22, 33, 50, 54). Interestingly, cytoplasmic expression of APE1 has been correlated with aggressiveness of different tumors (55, 56), although its role in tumorigenic processes is completely unknown. To date, no subnuclear distribution of APE1 has been reported. APE1 is an abundant protein (about 104 to 105 copies/cell) within eukaryotic cells and has a relatively long half-life (about 8 h). Thus, fine-tuning of the multiple APE1 functions may reside on its posttranslational modifications and on the modulation of its interactome under different conditions. While some posttranslational modifications have a functional role (i.e., Lys6/Lys7 acetylation) (7, 17), little information is available on APE1 protein interacting partners, except for those that are involved in BER function (38). Interestingly, proteolysis at residue Lys31 has recently been related to an enhanced immune cell death mediated by granzymes A and K (16, 23). This proteolytic event reduces APE1 accumulation within nuclei (16, 29) and its interaction with XRCC1 (60) and alters APE1 functions (16, 23). Recently, proteolysis occurring at Asn33 (giving rise to a protein form called NΔ33APE1) has also been described (11), suggesting that removal of the NLS may constitute a general mechanism for redirecting APE1 toward noncanonical subcellular compartments, such as mitochondria (11, 33, 54). Unfortunately, neither has the specific protease responsible for this cleavage been identified in nonimmune cells, nor has the mitochondrial localization signal been mapped yet. Mitochondrial localization of APE1 could be associated with a potential role in mtDNA repair of oxidized bases (11, 33, 54). However, since it is not clear whether NΔ33APE1 maintains its DNA repair activity in vivo (16) or acquires an aspecific endonuclease activity for double-stranded DNA (dsDNA) (66), at present it is not possible to drive any definitive conclusion. Moreover, as the truncated NΔ33APE1 form is associated with an apoptotic phenotype (23), it cannot be excluded that its generation may causatively be involved in the cytotoxic effect, driving proapoptotic triggering directly within mitochondria. The first 42 amino acids of APE1 are highly unordered in the protein crystallographic structure (3, 35), while the remainder of the protein has a globular fold (21). It is therefore presumable that the protein's N terminus is used for interacting with other partners, thus modulating the different APE1 functions. Interestingly, a similar bipartite arrangement for Rrp1, the Drosophila homologue of mammalian APE1, has been described, pointing out a functional role of the unstructured N-terminal domain in modulating protein-protein interactions (42, 52). By using an unbiased proteomic approach, in this work we have identified and characterized a novel APE1 complex. We found that APE1 N terminus is essential for binding to a number of proteins involved in ribosome biogenesis and RNA processing. Among the interacting partners, we focused on the nucleophosmin (NPM1)-APE1 interaction. NPM1 is an abundant protein which specifically resides within the granular region of the nucleolus and has been implicated in a variety of cellular processes, including centrosome duplication, maintenance of the genome's integrity, and ribosome biogenesis (19). NPM1 has a chaperone activity regulated by phosphorylation (51) and an endoribonuclease activity at a specific site of the spacer region between the 5.8S and the 28S rRNAs (43). Here, we demonstrate that the NPM1-APE1 interaction is required for APE1 subnuclear localization and for modulating the cleansing process of rRNA. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus possibly affecting gene expression through posttranscriptional mechanisms.

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.

bstract: 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, APE 1 /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 APE 1 /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 APE I /Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE 1 /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.

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. Analyses were performed on nine donor livers during LT. By using 2-DE and MALDI-TOF MS, we identified 36 proteins which resulted significantly altered upon I/R injury. The majority of these proteins are functionally involved in lipid and energy metabolism, in different metabolic pathways, in redox signalling and in oxidative-stress response. Our data represent the first global approach in the study of I/R injury in liver.

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.