Milena Romanello

APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process

ABSTRACT 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.

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.

Oxidative stress causes bone loss in estrogen-deficient mice through enhanced bone marrow dendritic cell activation

Increased production of tumor necrosis factor α (TNF) in the bone marrow (BM) in response to both oxidative stress and T cell activation contributes to the bone loss induced by estrogen deficiency, but it is presently unknown whether oxidative stress causes bone loss through T cells. Here we show that ovariectomy causes an accumulation in the BM of reactive oxygen species, which leads to increased production of TNF by activated T cells through up-regulation of the costimulatory molecule CD80 on dendritic cells. Accordingly, bone loss is prevented by treatment of ovariectomized mice with either antioxidants or CTLA4-Ig, an inhibitor of the CD80/CD28 pathway. In summary, reactive oxygen species accumulation in the BM is an upstream consequence of ovariectomy that leads to bone loss by activating T cells through enhanced activity of BM dendritic cells, and these findings suggest that the CD80/CD28 pathway may represent a therapeutic target for postmenopausal bone loss.

Connexin43 interacts with βarrestin: a pre-requisite for osteoblast survival induced by parathyroid hormone

Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP‐mediated signaling downstream of the G protein‐coupled receptor PTHR1. We present evidence herein that PTH‐induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock‐down abolished the expression of cAMP‐target genes and anti‐apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril‐cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of βarrestin, a PTHR1‐interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43Δ245) or unable to be phosphorylated at serine 368 (Cx43S368A), a residue crucial for Cx43 trafficking and function, failed to restore the anti‐apoptotic effect of PTH in Cx43‐deficient cells. In addition, overexpression of wild type βarrestin abrogated PTH survival signaling in Cx43‐expressing cells. Moreover, βarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43S368A; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43S368 phosphorylation or overexpression of wild type Cx43, but not Cx43Δ245 or Cx43S368A, reduced the interaction between βarrestin and the PTHR1. These studies demonstrate that βarrestin is a novel Cx43‐interacting protein and suggest that, by sequestering βarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH. J. Cell. Biochem. 112: 2920–2930, 2011.

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

The functional importance of APE1 nucleolar accumulation is described. It is shown that acetylation of Lys27–35, affecting local conformation, regulates APE1 function by 1) controlling its interaction with NPM1 and rRNA and its nucleolar accumulation, 2) modulating K6/K7 acetylation status, and 3) promoting APE1 BER activity in cells.

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

BackgroundNitrogen-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.ResultsWith 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.ConclusionsThe 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.

Functional characterization of the common c.-32-13T>G mutation of GAA gene: identification of potential therapeutic agents

Glycogen storage disease type II is a lysosomal storage disorder due to mutations of the GAA gene, which causes lysosomal alpha-glucosidase deficiency. Clinically, glycogen storage disease type II has been classified in infantile and late-onset forms. Most late-onset patients share the leaky splicing mutation c.-32-13T>G. To date, the mechanism by which the c.-32-13T>G mutation affects the GAA mRNA splicing is not fully known. In this study, we demonstrate that the c.-32-13T>G mutation abrogates the binding of the splicing factor U2AF65 to the polypyrimidine tract of exon 2 and that several splicing factors affect exon 2 inclusion, although the only factor capable of acting in the c.-32-13 T>G context is the SR protein family member, SRSF4 (SRp75). Most importantly, a preliminary screening using small molecules described to be able to affect splicing profiles, showed that resveratrol treatment resulted in a significant increase of normal spliced GAA mRNA, GAA protein content and activity in cells transfected with a mutant minigene and in fibroblasts from patients carrying the c-32-13T>G mutation. In conclusion, this work provides an in-depth functional characterization of the c.-32-13T>G mutation and, most importantly, an in vitro proof of principle for the use of small molecules to rescue normal splicing of c.-32-13T>G mutant alleles.

The glycosides of hydroxylysine are final products of collagen degradation in humans

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are metabolites derived from collagen degradation. They are useful biochemical markers provided they are not further processed. Experiments were run to test the activity of alpha- and beta-glycosidases in human kidney cortex preparations. Results allow to exclude the presence of the specific enzymes, in contrast with what is reported for the rat kidney tissue.

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.

The redox function of APE1 is involved in the differentiation process of stem cells toward a neuronal cell fate.

Low-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype. Methods and results: Applying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-κB, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation potential of stem cells towards specific neuronal subtypes. These findings provide a molecular basis for the redox-mediated hypothesis of neuronal differentiation program.