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1.  SIRT1 gene expression upon genotoxic damage is regulated by APE1 through nCaRE-promoter elements 
Molecular Biology of the Cell  2014;25(4):532-547.
APE1 is recruited to the transcription initiation site of the SIRT1 promoter during early cell response to oxidative stress. This reveals the importance of BER enzyme involvement in controlling specific gene expression at the transcriptional level.
Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein contributing to genome stability via repair of DNA lesions via the base excision repair pathway. It also plays a role in gene expression regulation and RNA metabolism. Another, poorly characterized function is its ability to bind to negative calcium responsive elements (nCaRE) of some gene promoters. The presence of many functional nCaRE sequences regulating gene transcription can be envisioned, given their conservation within ALU repeats. To look for functional nCaRE sequences within the human genome, we performed bioinformatic analyses and identified 57 genes potentially regulated by APE1. We focused on sirtuin-1 (SIRT1) deacetylase due to its involvement in cell stress, including senescence, apoptosis, and tumorigenesis, and its role in the deacetylation of APE1 after genotoxic stress. The human SIRT1 promoter presents two nCaRE elements stably bound by APE1 through its N-terminus. We demonstrate that APE1 is part of a multiprotein complex including hOGG1, Ku70, and RNA Pol II, which is recruited on SIRT1 promoter to regulate SIRT1 gene functions during early response to oxidative stress. These findings provide new insights into the role of nCaRE sequences in the transcriptional regulation of mammalian genes.
PMCID: PMC3923644  PMID: 24356447
2.  Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells 
PLoS ONE  2013;8(12):e84403.
Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line.
Methods and Principal Findings
We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis.
Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study were already known as TG2 substrates, we can also suppose that transamidating activity and differential phosphorylation of the same targets may represent a novel regulatory mechanism whose relevance in celiac disease pathogenesis is still unexplored.
PMCID: PMC3877280  PMID: 24391952
3.  Genome-wide analysis and proteomic studies reveal APE1/Ref-1 multifunctional role in mammalian cells 
Proteomics  2009;9(4):1058-1074.
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 to 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. Thus, 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.
PMCID: PMC3802553  PMID: 19180539
Cell signaling; Difference analysis; Transcription regulation; Two-dimensional gel electrophoresis; Microarrays; APE1; Ref-1; proteomics; siRNA; oxidative stress; apoptosis; mitochondria
4.  Transcriptomic and proteomic analysis of a compatible tomato-aphid interaction reveals a predominant salicylic acid-dependent plant response 
BMC Genomics  2013;14:515.
Aphids are among the most destructive pests in temperate climates, causing significant damage on several crops including tomato. We carried out a transcriptomic and proteomic study to get insights into the molecular mechanisms and dynamics of the tomato response to the Macrosyphum euphorbiae aphid.
The time course analysis of aphid infestation indicated a complex, dynamic pattern of gene expression. Several biological functions were affected and genes related to the stress and defence response were the most represented. The Gene Ontology categories of the differentially expressed genes (899) and identified proteins (57) indicated that the tomato response is characterized by an increased oxidative stress accompanied by the production of proteins involved in the detoxification of oxygen radicals. Aphids elicit a defense reaction based on the cross-communication of different hormone-related signaling pathways such as those related to the salicylic acid (SA), jasmonic acid (JA), ethylene and brassinosteroids. Among them, the SA-signaling pathway and stress-responsive SA-dependent genes play a dominant role. Furthermore, tomato response is characterized by a reduced accumulation of photosynthetic proteins and a modification of the expression of various cell wall related genes.
Our work allowed a more comprehensive understanding of the signaling events and the defense dynamics of the tomato response to aphids in a compatible interaction and, based on experimental data, a model of the tomato–aphid molecular interaction was proposed. Considering the rapid advancement of tomato genomics, this information will be important for the development of new protection strategies.
PMCID: PMC3733717  PMID: 23895395
Solanum lycopersicum; Macrosiphum euphorbiae; Plant-insect interactions; Defense; Salicylic acid; Jasmonic acid
5.  The proteome of Populus nigra woody root: response to bending 
Annals of Botany  2012;110(2):415-432.
Background and Aims
Morphological and biomechanical alterations occurring in woody roots of many plant species in response to mechanical stresses are well documented; however, little is known about the molecular mechanisms regulating these important alterations. The first forest tree genome to be decoded is that of Populus, thereby providing a tool with which to investigate the mechanisms controlling adaptation of woody roots to changing environments. The aim of this study was to use a proteomic approach to investigate the response of Populus nigra woody taproot to mechanical stress.
To simulate mechanical perturbations, the taproots of 30 one-year-old seedlings were bent to an angle of 90 ° using a steel net. A spatial and temporal two-dimensional proteome map of the taproot axis was obtained. We compared the events occurring in the above-bending, central bending and below-bending sectors of the taproot.
Key Results
The first poplar woody taproot proteome map is reported here; a total of 207 proteins were identified. Spatial and temporal proteomic analysis revealed that factors involved in plant defence, metabolism, reaction wood formation and lateral root development were differentially expressed in the various sectors of bent vs. control roots, seemingly in relation to the distribution of mechanical forces along the stressed woody taproots. A complex interplay among different signal transduction pathways involving reactive oxygen species appears to modulate these responses.
Poplar woody root uses different temporal and spatial mechanisms to respond to mechanical stress. Long-term bending treatment seem to reinforce the defence machinery, thereby enabling the taproot to better overcome winter and to be ready to resume growth earlier than controls.
PMCID: PMC3394638  PMID: 22437664
Growth cycle; mechanical stress; Populus nigra; proteomics; proteome; roots
6.  Synthesis and Characterization of the 47-Residue Heterodimeric Antimicrobial Peptide Distinctin, Featuring Directed Disulfide Bridge Formation 
Biopolymers  2012;98(5):479-484.
Distinctin, a 47-residue heterodimeric peptide with potent antimicrobial activity, comprises two monomeric units linked covalently by a disulfide bond between Cys19 from the 22-residue A chain and Cys23 from the 25-residue B chain. Previous synthetic strategies involved assemblies of the two individual chains, followed by their co-oxidation to form the connecting disulfide bridge, and resulted in a mixture of three species: two homodimers and one heterodimer. Here, we report synthesis of exclusively heterodimeric distinctin, using recently developed tactics for directed disulfide bridge formation. Material prepared this way was characterized and found to be suitable for more detailed structural studies.
PMCID: PMC3551542  PMID: 23203692
Directed disulfide bridge formation; solid-phase synthesis; distinctin; antimicrobial peptides; solution NMR
7.  Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells 
Molecular Biology of the Cell  2012;23(20):4079-4096.
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.
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 (K27/K31/K32/K35) 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 K27/K31/K32/K35 modulates acetylation at K6/K7 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 K27/K31/K32/K35 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.
PMCID: PMC3469522  PMID: 22918947
8.  Adaptative biochemical pathways and regulatory networks in Klebsiella oxytoca BAS-10 producing a biotechnologically relevant exopolysaccharide during Fe(III)-citrate fermentation 
A bacterial strain previously isolated from pyrite mine drainage and named BAS-10 was tentatively identified as Klebsiella oxytoca. Unlikely other enterobacteria, BAS-10 is able to grow on Fe(III)-citrate as sole carbon and energy source, yielding acetic acid and CO2 coupled with Fe(III) reduction to Fe(II) and showing unusual physiological characteristics. In fact, under this growth condition, BAS-10 produces an exopolysaccharide (EPS) having a high rhamnose content and metal-binding properties, whose biotechnological applications were proven as very relevant.
Further phylogenetic analysis, based on 16S rDNA sequence, definitively confirmed that BAS-10 belongs to K. oxytoca species. In order to rationalize the biochemical peculiarities of this unusual enterobacteriun, combined 2D-Differential Gel Electrophoresis (2D-DIGE) analysis and mass spectrometry procedures were used to investigate its proteomic changes: i) under aerobic or anaerobic cultivation with Fe(III)-citrate as sole carbon source; ii) under anaerobic cultivations using Na(I)-citrate or Fe(III)-citrate as sole carbon source. Combining data from these differential studies peculiar levels of outer membrane proteins, key regulatory factors of carbon and nitrogen metabolism and enzymes involved in TCA cycle and sugar biosynthesis or required for citrate fermentation and stress response during anaerobic growth on Fe(III)-citrate were revealed. The protein differential regulation seems to ensure efficient cell growth coupled with EPS production by adapting metabolic and biochemical processes in order to face iron toxicity and to optimize energy production.
Differential proteomics provided insights on the molecular mechanisms necessary for anaeorobic utilization of Fe(III)-citrate in a biotechnologically promising enterobacteriun, also revealing genes that can be targeted for the rational design of high-yielding EPS producer strains.
PMCID: PMC3539929  PMID: 23176641
9.  Molecular interactions between the olive and the fruit fly Bactrocera oleae 
BMC Plant Biology  2012;12:86.
The fruit fly Bactrocera oleae is the primary biotic stressor of cultivated olives, causing direct and indirect damages that significantly reduce both the yield and the quality of olive oil. To study the olive-B. oleae interaction, we conducted transcriptomic and proteomic investigations of the molecular response of the drupe. The identifications of genes and proteins involved in the fruit response were performed using a Suppression Subtractive Hybridisation technique and a combined bi-dimensional electrophoresis/nanoLC-ESI-LIT-MS/MS approach, respectively.
We identified 196 ESTs and 26 protein spots as differentially expressed in olives with larval feeding tunnels. A bioinformatic analysis of the identified non-redundant EST and protein collection indicated that different molecular processes were affected, such as stress response, phytohormone signalling, transcriptional control and primary metabolism, and that a considerable proportion of the ESTs could not be classified. The altered expression of 20 transcripts was also analysed by real-time PCR, and the most striking differences were further confirmed in the fruit of a different olive variety. We also cloned the full-length coding sequences of two genes, Oe-chitinase I and Oe-PR27, and showed that these are wound-inducible genes and activated by B. oleae punctures.
This study represents the first report that reveals the molecular players and signalling pathways involved in the interaction between the olive fruit and its most damaging biotic stressor. Drupe response is complex, involving genes and proteins involved in photosynthesis as well as in the production of ROS, the activation of different stress response pathways and the production of compounds involved in direct defence against phytophagous larvae. Among the latter, trypsin inhibitors should play a major role in drupe resistance reaction.
PMCID: PMC3733423  PMID: 22694925
Olea europea; Pest; SSH; Proteomics; Defence; Fruit fly
10.  Proteomic Characterization of a Mouse Model of Familial Danish Dementia 
A dominant mutation in the ITM2B/BRI2 gene causes familial Danish dementia (FDD) in humans. To model FDD in animal systems, a knock-in approach was recently implemented in mice expressing a wild-type and mutant allele, which bears the FDD-associated mutation. Since these FDDKI mice show behavioural alterations and impaired synaptic function, we characterized their synaptosomal proteome via two-dimensional differential in-gel electrophoresis. After identification by nanoliquid chromatography coupled to electrospray-linear ion trap tandem mass spectrometry, the differentially expressed proteins were classified according to their gene ontology descriptions and their predicted functional interactions. The Dlg4/Psd95 scaffold protein and additional signalling proteins, including protein phosphatases, were revealed by STRING analysis as potential players in the altered synaptic function of FDDKI mice. Immunoblotting analysis finally demonstrated the actual downregulation of the synaptosomal scaffold protein Dlg4/Psd95 and of the dual-specificity phosphatase Dusp3 in the synaptosomes of FDDKI mice.
PMCID: PMC3350990  PMID: 22619496
11.  Probing Membrane Topology of the Antimicrobial Peptide Distinctin by Solid-State NMR Spectroscopy in Zwitterionic and Charged Lipid Bilayers 
Biochimica et biophysica acta  2010;1808(1):34-40.
Distinctin is a 47-residue antimicrobial peptide, which interacts with negatively charged membranes and is active against Gram-positive and Gram-negative bacteria. Its primary sequence comprises two linear chains of 22 (chain 1) and 25 (chain 2) residues, linked by a disulfide bridge between Cys19 of chain 1 and Cys23 of chain 2. Unlike other antimicrobial peptides, distinctin in the absence of the lipid membrane has a well-defined three-dimensional structure, which protects it from protease degradation. Here, we used static solid-state NMR spectroscopy to study the topology of distinctin in lipid bilayers. We found that In mechanically aligned lipid bilayers (charged or zwitterionic) this heterodimeric peptide adopts an ordered conformation absorbed on the surface of the membrane, with the long helix (chain 2), approximately parallel to the lipid bilayer (∼5° from the membrane plane) and the short helix (chain 1) forming a ∼24° angle. Since at lipid-to-protein molar ratio of 50:1 the peptide does not disrupt the macroscopic alignment of either charged or zwitterionic lipid bilayers, it is possible that higher concentrations might be needed for the hypothesized pore formation, or alternatively, distinctin elicits its cell disruption action by other mechanisms.
PMCID: PMC2997851  PMID: 20719234
12.  Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function 
Molecular Biology of the Cell  2011;22(20):3887-3901.
The multifunctional APE1 protein is required for tumor progression and is associated with cancer resistance. It is shown that APE1 presents structural elements that function in distinct cellular roles, highlighting the molecular determinants of the multifunctional nature of this protein and providing the basis for a new role of the C65 residue.
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.
PMCID: PMC3192867  PMID: 21865600
13.  Proteomic Analysis of Sera from Common Variable Immunodeficiency Patients Undergoing Replacement Intravenous Immunoglobulin Therapy 
Common variable immunodeficiency is the most common form of symptomatic primary antibody failure in adults and children. Replacement immunoglobulin is the standard treatment of these patients. By using a differential proteomic approach based on 2D-DIGE, we examined serum samples from normal donors and from matched, naive, and immunoglobulin-treated patients. The results highlighted regulated expression of serum proteins in naive patients. Among the identified proteins, clusterin/ApoJ serum levels were lower in naive patients, compared to normal subjects. This finding was validated in a wider collection of samples from newly enrolled patients. The establishment of a cellular system, based on a human hepatocyte cell line HuH7, allowed to ascertain a potential role in the regulation of CLU gene expression by immunoglobulins.
PMCID: PMC3180879  PMID: 21960740
14.  BRCA1 modulates the expression of hnRNPA2B1 and KHSRP 
Cell Cycle  2010;9(23):4666-4673.
Inactivation of the breast cancer susceptibility gene 1 (BRCA1) plays a significant role in the development of a subset of familial breast and ovarian cancers, but increasing evidence points to a role also in sporadic tumors. BRCA1 is a multifunctional nuclear protein involved in the regulation of many nuclear cellular processes, including DNA repair, cell cycle, transcription and chromatin remodeling. To identify novel proteins participating in the BRCA1 network, two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry were used to compare the nuclear-enriched proteome map of BRCA1-deficient and BRCA1-proficient cell lines. Five differentially expressed polypeptides were identified and two of them, hnRNPA2B1 and KHSRP, turned out to be involved in mRNA and miRNA metabolism. qRT-PCR analyses indicated that the hnRNPA2B1 and KHSRP levels increased in response to BRCA1 loss and restoration of BRCA1 expression in BRCA1 null cells reverted hnRNPA2B1 and KHSRP upregulation. Interrogation of publicly available transcriptional profiling datasets revealed that both genes were actually overexpressed in BRCA1 mutated tumors. Overall, our results indicate that BRCA1 modulates the expression of two proteins involved in the processing of RNA, highlighting the complex nature of BRCA1-associated tumor suppressor function and disclosing a novel mechanism by which BRCA1 may affect transcription.
PMCID: PMC3048036  PMID: 21099359
BRCA1; proteomics; hnRNPA2B1; KHSRP; RNA metabolism
15.  Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations 
Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted.
Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38 ± 0.02 and 0.33 ± 0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA).
In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement.
PMCID: PMC3004843  PMID: 21110849
16.  Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions 
Nucleic Acids Research  2010;38(22):8239-8256.
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.
PMCID: PMC3001066  PMID: 20699270
17.  APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process ▿ †  
Molecular and Cellular Biology  2009;29(7):1834-1854.
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.
PMCID: PMC2655621  PMID: 19188445
18.  Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge 
Exposure to nickel (Ni) and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress.
To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts.
These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.
PMCID: PMC1995220  PMID: 17692131
19.  Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro 
Molecular and Cellular Biology  2002;22(19):6663-6668.
Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2′-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D′ of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-l-methionine-binding region of Nop1p is responsible for the catalytic activity.
PMCID: PMC134041  PMID: 12215523

Results 1-19 (19)