Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Role of condensates in modulating DNA repair pathways and its implication for chemoresistance.

For cells, it is important to repair DNA damage, such as double-strand and single-strand DNA breaks, because unrepaired DNA can compromise genetic integrity, potentially leading to cell death or cancer. Cells have multiple DNA damage repair pathways that have been the subject of detailed genetic, biochemical, and structural studies. Recently, the scientific community has started to gain evidence that the repair of DNA double-strand breaks may occur within biomolecular condensates and that condensates may also contribute to DNA damage through concentrating genotoxic agents used to treat various cancers. Here, we summarize key features of biomolecular condensates and note where they have been implicated in the repair of DNA double-strand breaks. We also describe evidence suggesting that condensates may be involved in the repair of other types of DNA damage, including single-strand DNA breaks, nucleotide modifications (e.g., mismatch and oxidized bases), and bulky lesions, among others. Finally, we discuss old and new mysteries that could now be addressed considering the properties of condensates, including chemoresistance mechanisms.

A regulatory network comprising let-7 miRNA and SMUG1 is associated with good prognosis in ER+ breast tumours.

Single-strand selective uracil-DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.

APE1 controls DICER1 expression in NSCLC through miR-33a and miR-130b.

Increasing evidence suggests different, not completely understood roles of microRNA biogenesis in the development and progression of lung cancer. The overexpression of the DNA repair protein apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an important cause of poor chemotherapeutic response in lung cancer and its involvement in onco-miRNAs biogenesis has been recently described. Whether APE1 regulates miRNAs acting as prognostic biomarkers of lung cancer has not been investigated, yet. In this study, we analyzed miRNAs differential expression upon APE1 depletion in the A549 lung cancer cell line using high-throughput methods. We defined a signature of 13 miRNAs that strongly correlate with APE1 expression in human lung cancer: miR-1246, miR-4488, miR-24, miR-183, miR-660, miR-130b, miR-543, miR-200c, miR-376c, miR-218, miR-146a, miR-92b and miR-33a. Functional enrichment analysis of this signature revealed its biological relevance in cancer cell proliferation and survival. We validated DICER1 as a direct functional target of the APE1-regulated miRNA-33a-5p and miR-130b-3p. Importantly, IHC analyses of different human tumors confirmed a negative correlation existing between APE1 and Dicer1 protein levels. DICER1 downregulation represents a prognostic marker of cancer development but the mechanisms at the basis of this phenomenon are still completely unknown. Our findings, suggesting that APE1 modulates DICER1 expression via miR-33a and miR-130b, reveal new mechanistic insights on DICER1 regulation, which are of relevance in lung cancer chemoresistance and cancer invasiveness.

Enzymatically active apurinic/apyrimidinic endodeoxyribonuclease 1 is released by mammalian cells through exosomes.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.

Combining Deep Phenotyping of Serum Proteomics and Clinical Data via Machine Learning for COVID-19 Biomarker Discovery.

The persistence of long-term coronavirus-induced disease 2019 (COVID-19) sequelae demands better insights into its natural history. Therefore, it is crucial to discover the biomarkers of disease outcome to improve clinical practice. In this study, 160 COVID-19 patients were enrolled, of whom 80 had a "non-severe" and 80 had a "severe" outcome. Sera were analyzed by proximity extension assay (PEA) to assess 274 unique proteins associated with inflammation, cardiometabolic, and neurologic diseases. The main clinical and hematochemical data associated with disease outcome were grouped with serological data to form a dataset for the supervised machine learning techniques. We identified nine proteins (i.e., CD200R1, MCP1, MCP3, IL6, LTBP2, MATN3, TRANCE, α2-MRAP, and KIT) that contributed to the correct classification of COVID-19 disease severity when combined with relative neutrophil and lymphocyte counts. By analyzing PEA, clinical and hematochemical data with statistical methods that were able to handle many variables in the presence of a relatively small sample size, we identified nine potential serum biomarkers of a "severe" outcome. Most of these were confirmed by literature data. Importantly, we found three biomarkers associated with central nervous system pathologies and protective factors, which were downregulated in the most severe cases.

Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination.

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.

Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA.

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.

New perspectives in cancer biology from a study of canonical and non-canonical functions of base excision repair proteins with a focus on early steps.

Alterations of DNA repair enzymes and consequential triggering of aberrant DNA damage response (DDR) pathways are thought to play a pivotal role in genomic instabilities associated with cancer development, and are further thought to be important predictive biomarkers for therapy using the synthetic lethality paradigm. However, novel unpredicted perspectives are emerging from the identification of several non-canonical roles of DNA repair enzymes, particularly in gene expression regulation, by different molecular mechanisms, such as (i) non-coding RNA regulation of tumour suppressors, (ii) epigenetic and transcriptional regulation of genes involved in genotoxic responses and (iii) paracrine effects of secreted DNA repair enzymes triggering the cell senescence phenotype. The base excision repair (BER) pathway, canonically involved in the repair of non-distorting DNA lesions generated by oxidative stress, ionising radiation, alkylation damage and spontaneous or enzymatic deamination of nucleotide bases, represents a paradigm for the multifaceted roles of complex DDR in human cells. This review will focus on what is known about the canonical and non-canonical functions of BER enzymes related to cancer development, highlighting novel opportunities to understand the biology of cancer and representing future perspectives for designing new anticancer strategies. We will specifically focus on APE1 as an example of a pleiotropic and multifunctional BER protein.

Differential capacity of CD90+ cells in autophagy activation following chemotherapy in hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Analysis of cancer biomarkers is an important tool in developing targeted-therapy and in modulating chemoresistance. Here, we analyze the relevance of CD90, a marker of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) and its correlation with autophagy. MATERIALS AND METHODS: For in vivo study, 86 specimens were collected from 43 patients undergoing liver resections. In each patient, HCC nodule (HCC) and surrounding non-tumor (SNT) were collected. For in vitro study, HCC cells JHH6 subpopulations expressing CD90+ and CD90- were isolated using magnetic-sorter and confirmed by flow-cytometry. Upon doxorubicin treatment, autophagy turn-over was analyzed by RTqPCR for mRNA expression, Western blot for protein expression, and autophagosome staining for autophagy-flux. Cytotoxicity test was performed by MTT assay. Gene and protein analysis were performed in clinical samples together with immunohistostaining. RESULTS: CD90 mRNA expression was higher in HCC than in SNT for 8-fold (p < 0.001). LC3-II protein was up-regulated in the HCC in comparison with the SNT (p < 0.05). In vitro model showed that CD90+ and CD90- cells had diverse expressions of autophagy-related genes. Upon doxorubicin treatment, autophagy was activated in both cells by increasing LC3-II protein expression, autophagic vacuoles, and dysregulation of autophagy-related mRNAs. A differential autophagic capacity was noticed between two subpopulations and it was correlated with cellular toxicity assay. CONCLUSIONS: We demonstrated the relevance of differential autophagy capacity of CD90+ cells in HCC. Autophagy was involved in cancer-defense mechanism against doxorubicin. Cancer promoting function of autophagy in CD90+ cells was also related to cancer environment.

Somatostatin as Inflow Modulator in Liver-transplant Recipients With Severe Portal Hypertension: A Randomized Trial.

OBJECTIVE: To investigate the safety and efficacy of somatostatin as liver inflow modulator in patients with end-stage liver disease (ESLD) and clinically significant portal hypertension (CSPH) undergoing liver transplantation (LT) (ClinicalTrials.gov number,01290172). BACKGROUND: In LT, portal hyperperfusion can severely impair graft function and survival, mainly in cases of partial LT. METHODS: Thirty-three patients undergoing LT for ESLD and CSPH were randomized double-blindly to receive somatostatin or placebo (2:1). The study drug was administered intraoperatively as 5-mL bolus (somatostatin: 500 µg), followed by a 2.5 mL/h infusion (somatostatin: 250 µg/h) for 5 days. Hepatic and systemic hemodynamics were measured, along with liver function tests and clinical outcomes. The ischemia-reperfusion injury (IRI) was analyzed through histological and protein expression analysis. RESULTS: Twenty-nine patients (18 receiving somatostatin, 11 placebo) were included in the final analysis. Ten patients responded to somatostatin bolus, with a significant decrease in hepatic venous portal gradient (HVPG) and portal flow of -28.3% and -29.1%, respectively. At graft reperfusion, HVPG was lower in patients receiving somatostatin (-81.7% vs -58.8%; P = 0.0084), whereas no difference was observed in the portal flow (P = 0.4185). Somatostatin infusion counteracted the decrease in arterial flow (-10% vs -45%; P = 0.0431). There was no difference between the groups in the severity of IRI, incidence of adverse events, long-term complications, graft, and patient survival. CONCLUSIONS: Somatostatin infusion during LT in patients with CSPH is safe, reduces the HVPG, and preserves the arterial inflow to the graft. This study establishes the efficacy of somatostatin as a liver inflow modulator.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2.

Ribonucleoside 5'-monophosphates (rNMPs) are the most common non-standard nucleotides found in DNA of eukaryotic cells, with over 100 million rNMPs transiently incorporated in the mammalian genome per cell cycle. Human ribonuclease (RNase) H2 is the principal enzyme able to cleave rNMPs in DNA. Whether RNase H2 may process abasic or oxidized rNMPs incorporated in DNA is unknown. The base excision repair (BER) pathway is mainly responsible for repairing oxidized and abasic sites into DNA. Here we show that human RNase H2 is unable to process an abasic rNMP (rAP site) or a ribose 8oxoG (r8oxoG) site embedded in DNA. On the contrary, we found that recombinant purified human apurinic/apyrimidinic endonuclease-1 (APE1) and APE1 from human cell extracts efficiently process an rAP site in DNA and have weak endoribonuclease and 3'-exonuclease activities on r8oxoG substrate. Using biochemical assays, our results provide evidence of a human enzyme able to recognize and process abasic and oxidized ribonucleotides embedded in DNA.

Platinum Salts in Patients with Breast Cancer: A Focus on Predictive Factors.

Breast cancer (BC) is the most frequent oncologic cause of death among women and the improvement of its treatments is compelling. Platinum salts (e.g., carboplatin, cisplatin, and oxaliplatin) are old drugs still used to treat BC, especially the triple-negative subgroup. However, only a subset of patients see a concrete benefit from these drugs, raising the question of how to select them properly. Therefore, predictive biomarkers for platinum salts in BC still represent an unmet clinical need. Here, we review clinical and preclinical works in order to summarize the current evidence about predictive or putative platinum salt biomarkers in BC. The association between BRCA1/2 gene mutations and platinum sensitivity has been largely described. However, beyond the mutations of these two genes, several other proteins belonging to the homologous recombination pathways have been linked to platinum response, defining the concept of BRCAness. Several works, here reviewed, have tried to capture BRCAness through different strategies, such as homologous recombination deficiency (HRD) score and genetic signatures. Moreover, p53 and its family members (p63 and p73) might also be used as predictors of platinum response. Finally, we describe the mounting preclinical evidence regarding base excision repair deficiency as a possible new platinum biomarker.

Mitochondrial translocation of APE1 relies on the MIA pathway.

APE1 is a multifunctional protein with a fundamental role in repairing nuclear and mitochondrial DNA lesions caused by oxidative and alkylating agents. Unfortunately, comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary and contrasting. Recent data demonstrate that APE1 interacts with the mitochondrial import and assembly protein Mia40 suggesting the involvement of a redox-assisted mechanism, dependent on the disulfide transfer system, to be responsible of APE1 trafficking into the mitochondria. The MIA pathway is an import machinery that uses a redox system for cysteine enriched proteins to drive them in this compartment. It is composed by two main proteins: Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 after the import. In this study, we demonstrated that: (i) APE1 and Mia40 interact through disulfide bond formation; and (ii) Mia40 expression levels directly affect APE1's mitochondrial translocation and, consequently, play a role in the maintenance of mitochondrial DNA integrity. In summary, our data strongly support the hypothesis of a redox-assisted mechanism, dependent on Mia40, in controlling APE1 translocation into the mitochondrial inner membrane space and thus highlight the role of this protein transport pathway in the maintenance of mitochondrial DNA stability and cell survival.

The prion protein is critical for DNA repair and cell survival after genotoxic stress.

The prion protein (PrP) is highly conserved and ubiquitously expressed, suggesting that it plays an important physiological function. However, despite decades of investigation, this role remains elusive. Here, by using animal and cellular models, we unveil a key role of PrP in the DNA damage response. Exposure of neurons to a genotoxic stress activates PRNP transcription leading to an increased amount of PrP in the nucleus where it interacts with APE1, the major mammalian endonuclease essential for base excision repair, and stimulates its activity. Preventing the induction of PRNP results in accumulation of abasic sites in DNA and impairs cell survival after genotoxic treatment. Brains from Prnp(-/-) mice display a reduced APE1 activity and a defect in the repair of induced DNA damage in vivo. Thus, PrP is required to maintain genomic stability in response to genotoxic stresses.

Isoforms of the Erythropoietin receptor in dopaminergic neurons of the Substantia Nigra.

Erythropoietin receptor (EpoR) regulates erythrocytes differentiation in blood. In the brain, EpoR has been shown to protect several neuronal cell types from cell death, including the A9 dopaminergic neurons (DA) of the Substantia Nigra (SN). These cells form the nigrostriatal pathway and are devoted to the control of postural reflexes and voluntary movements. Selective degeneration of A9 DA neurons leads to Parkinson's disease. By the use of nanoCAGE, a technology that allows the identification of Transcription Start Sites (TSSs) at a genome-wide level, we have described the promoter-level expression atlas of mouse A9 DA neurons purified with Laser Capture Microdissection (LCM). Here, we identify mRNA variants of the Erythropoietin Receptor (DA-EpoR) transcribed from alternative TSSs. Experimental validation and full-length cDNA cloning is integrated with gene expression analysis in the FANTOM5 database. In DA neurons, the EpoR gene encodes for a N-terminal truncated receptor. Based on STAT5 phosphorylation assays, we show that the new variant of N-terminally truncated EpoR acts as decoy when co-expressed with the full-length form. A similar isoform is also found in human. This work highlights new complexities in the regulation of Erythropoietin (EPO) signaling in the brain.

Inhibitors of the apurinic/apyrimidinic endonuclease 1 (APE1)/nucleophosmin (NPM1) interaction that display anti-tumor properties.

The apurinic/apyrimidinic endonuclease 1 (APE1) is a protein central to the base excision DNA repair pathway and operates in the modulation of gene expression through redox-dependent and independent mechanisms. Aberrant expression and localization of APE1 in tumors are recurrent hallmarks of aggressiveness and resistance to therapy. We identified and characterized the molecular association between APE1 and nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability and rRNA maturation. This protein-protein interaction modulates subcellular localization and endonuclease activity of APE1. Moreover, we reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, with NPM1 overexpression being a marker of poor prognosis. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Therefore, targeting the APE1/NPM1 interaction might represent an innovative strategy for the development of anticancer drugs, as tumor cells relying on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than untransformed cells. We set up a chemiluminescence-based high-throughput screening assay in order to find small molecules able to interfere with the APE1/NPM1 interaction. This screening led to the identification of a set of bioactive compounds that impair the APE1/NPM1 association in living cells. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to therapeutically relevant genotoxins. Given the prognostic significance of APE1 and NPM1, these compounds might prove effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas.

Placental aging and oxidation damage in a tissue micro-array model: an immunohistochemistry study.

To evaluate the expression of markers correlated with cellular senescence and DNA damage (8-hydroxy-2'-deoxy-guanosine (8-OHdG), p53, p21, APE1/Ref-1 (APE1), interleukin (IL-6 and IL-8) in placentas from healthy and pathologic pregnancies. This retrospective study considered a placental tissue micro-array containing 92 controls from different gestational ages and 158 pathological cases including preeclampsia (PE), HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count), small for gestational age (SGA) fetuses, and intrauterine growth restriction (IUGR) occurring at different gestational ages. In this study, we demonstrated a significant influence of gestational age on the expression in the trophoblast of 8-OHdG, p53, p21, APE1, and IL-6. In placentas of cases affected by PE, HELLP, or IUGR, there was an increased expression of 8-OHdG, p53, APE1, and IL-6 compared to controls (only IL-8 was significantly decreased in cases). In both groups of pathology between 22- and 34-week gestation and after 34-week gestation, APE1 levels were higher in the trophoblast of women affected by hypertensive disorders of pregnancy than women carrying an IUGR fetus. The cytoplasmic expression of 8-OHdG was increased in placentas in IUGR cases compared to PE or HELLP pregnancies. In cases after 34-week gestation, p21 was higher in SGA and IUGR than in controls and late PE. Moreover, p53 was increased after 34-week gestation in IUGR pregnancies. Placentas from pathological pregnancies had an altered expression of 8-OHdG, p53, p21, APE1, IL-6, and IL-8. The alterations of intracellular pathways involving these elements may be the cause or the consequence of placental dysfunction, but in any case reflect an impaired placental function, possibly due to increased aging velocity in pathologic cases.

G-quadruplex DNA recognition by nucleophosmin: new insights from protein dissection.

BACKGROUND: Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region. METHODS: Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif. RESULTS: These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability. CONCLUSIONS: These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif. GENERAL OBSERVATION: This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.

Role of the unstructured N-terminal domain of the hAPE1 (human apurinic/apyrimidinic endonuclease 1) in the modulation of its interaction with nucleic acids and NPM1 (nucleophosmin).

The hAPE1 (human apurinic/apyrimidinic endonuclease 1) is an essential enzyme, being the main abasic endonuclease in higher eukaryotes. However, there is strong evidence to show that hAPE1 can directly bind specific gene promoters, thus modulating their transcriptional activity, even in the absence of specific DNA damage. Recent findings, moreover, suggest a role for hAPE1 in RNA processing, which is modulated by the interaction with NPM1 (nucleophosmin). Independent domains account for many activities of hAPE1; however, whereas the endonuclease and the redox-active portions of the protein are well characterized, a better understanding of the role of the unstructured N-terminal region is needed. In the present study, we characterized the requirements for the interaction of hAPE1 with NPM1 and undamaged nucleic acids. We show that DNA/RNA secondary structure has an impact on hAPE1 binding in the absence of damage. Biochemical studies, using the isolated N-terminal region of the protein, reveal that the hAPE1 N-terminal domain represents an evolutionary gain of function, since its composition affects the protein's stability and ability to interact with both nucleic acids and NPM1. Although required, however, this region is not sufficient itself to stably interact with DNA or NPM1.