Pietro Pucci

Professor of Biochemistry

Name Pietro
Surname Pucci
Institution Università degli Studi della Campania Luigi Vanvitelli
Telephone +39 081 674 318 (UniNa)
Telephone 2 +39 081 373 7896 (Ceinge)
E-Mail pucci@unina.it
Address Department of Chemical Sciences, Federico II University, Via Cintia 6, 80126, Naples, Italy
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Pietro Pucci


  • cis-acting sequences and trans-acting factors in the localization of mRNA for mitochondrial ribosomal proteins.

    Publication Date: 01/12/2008 on Biochimica et biophysica acta
    by Russo A, Cirulli C, Amoresano A, Pucci P, Pietropaolo C, Russo G
    DOI: 10.1016/j.bbagrm.2008.08.006

    mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. Although several mechanisms have been identified, emerging evidence suggests that most transcripts reach the protein functional site by moving along cytoskeleton elements. We demonstrated previously that mRNA for mitochondrial ribosomal proteins are asymmetrically distributed in the cytoplasm, and that localization in the proximity of mitochondria is mediated by the 3'-UTR. Here we show by biochemical analysis that these mRNA transcripts are associated with the cytoskeleton through the microtubule network. Cytoskeleton association is functional for their intracellular localization near the mitochondrion, and the 3'-UTR is involved in this cytoskeleton-dependent localization. To identify the minimal elements required for localization, we generated DNA constructs containing, downstream from the GFP gene, deletion mutants of mitochondrial ribosomal protein S12 3'-UTR, and expressed them in HeLa cells. RT-PCR analysis showed that the localization signals responsible for mRNA localization are located in the first 154 nucleotides. RNA pull-down assays, mass spectrometry, and RNP immunoprecipitation assay experiments, demonstrated that mitochondrial ribosomal protein S12 3'-UTR interacts specifically with TRAP1 (tumor necrosis factor receptor-associated protein1), hnRNPM4 (heterogeneous nuclear ribonucleoprotein M4), Hsp70 and Hsp60 (heat shock proteins 70 and 60), and alpha-tubulin in vitro and in vivo.

  • In HspA from Helicobacter pylori vicinal disulfide bridges are a key determinant of domain B structure.

    Publication Date: 15/10/2008 on FEBS letters
    by Loguercio S, Dian C, Flagiello A, Scannella A, Pucci P, Terradot L, Zagari A
    DOI: 10.1016/j.febslet.2008.09.025

    Helicobacter pylori produces a heat shock protein A (HspA) that is unique to this bacteria. While the first 91 residues (domain A) of the protein are similar to GroES, the last 26 (domain B) are unique to HspA. Domain B contains eight histidines and four cysteines and was suggested to bind nickel. We have produced HspA and two mutants: Cys94Ala and Cys94Ala/Cys111Ala and identified the disulfide bridge pattern of the protein. We found that the cysteines are engaged in three disulfide bonds: Cys51/Cys53, Cys94/Cys111 and Cys95/Cys112 that result in a unique closed loop structure for the domain B.

  • The different forms of PNS myelin P0 protein within and outside lipid rafts.

    Publication Date: 01/10/2008 on Journal of neurochemistry
    by Fasano A, Amoresano A, Rossano R, Carlone G, Carpentieri A, Liuzzi GM, Pucci P, Riccio P
    DOI: 10.1111/j.1471-4159.2008.05598.x

    It is now well established that plasma membranes, such as the myelin sheath, are made of different microdomains with different lipid and protein composition. Lipid rafts are made mainly of sphingolipids and cholesterol, whereas the non-raft regions are made mainly of phosphoglycerides. Most myelin proteins may distribute themselves in raft and non-raft microdomains but the driving force that gives rise to their different distribution is not known yet. In this paper, we have studied the distribution of protein zero (P0), the most representative protein of PNS myelin, in the membrane microdomains. To this end, we have purified P0 from both non-raft (soluble P0, P0-S) and raft (P0-R) regions of PNS. Purified proteins were analyzed by two-dimensional gel electrophoresis and identified and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A detailed structural description of the two P0 forms is given in terms of amino acid sequence, post-translational modifications, and composition of associated lipids. Our findings suggest that structural differences between the two proteins, mainly related to the glycogroups, might be responsible for their different localization.

  • Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44.

    Publication Date: 01/09/2008 on Human molecular genetics
    by Fraldi A, Zito E, Annunziata F, Lombardi A, Cozzolino M, Monti M, Spampanato C, Ballabio A, Pucci P, Sitia R, Cosma MP
    DOI: 10.1093/hmg/ddn161

    Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple sulfatase deficiency, a rare recessive disorder in which all sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion.

  • Protease treatment affects both invasion ability and biofilm formation in Listeria monocytogenes.

    Publication Date: 01/07/2008 on Microbial pathogenesis
    by Longhi C, Scoarughi GL, Poggiali F, Cellini A, Carpentieri A, Seganti L, Pucci P, Amoresano A, Cocconcelli PS, Artini M, Costerton JW, Selan L
    DOI: 10.1016/j.micpath.2008.01.007

    Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-borne disease in humans. Several surface proteins have been shown to be essential in the adhesion of L. monocytogenes, and in the subsequent invasion of phagocytes. Because the control of the invasion of host cells by Listeria could potentially hinder its spread in the infected host, we have examined the effects of a protease treatment on the ability of L. monocytogenes to form biofilms and to invade tissues. We have chosen serratiopeptidase (SPEP), an extracellular metalloprotease produced by Serratia marcescens that is already widely used as an anti-inflammatory agent, and has been shown to modulate adhesin expression and to induce antibiotic sensitivity in other bacteria. Treatment of L. monocytogenes with sublethal concentrations of SPEP reduced their ability to form biofilms and to invade host cells. Zymograms of the treated cells revealed that Ami4b autolysin, internalinB, and ActA were sharply reduced. These cell-surface proteins are known to function as ligands in the interaction between these bacteria and their host cells, and our data suggest that treatment with this natural enzyme may provide a useful tool in the prevention of the initial adhesion of L. monocytogenes to the human gut.

  • Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity.

    Publication Date: 01/05/2008 on Chemistry & biology
    by Erbs G, Silipo A, Aslam S, De Castro C, Liparoti V, Flagiello A, Pucci P, Lanzetta R, Parrilli M, Molinaro A, Newman MA, Cooper RM
    DOI: 10.1016/j.chembiol.2008.03.017

    Peptidoglycan (PGN) is a unique and essential structural part of the bacterial cell wall. PGNs from two contrasting Gram-negative plant pathogenic bacteria elicited components characteristic of the innate immune system in Arabidopsis thaliana, such as transcription of the defense gene PR1, oxidative burst, medium alkalinization, and formation of callose. Highly purified muropeptides from PGNs were more effective elicitors of early defense responses than native PGN. Therefore, PGN and its constituents represent a Microbe-Associated Molecular Pattern (MAMP) in plant-bacterial interactions. PGN and muropeptides from aggressive Xanthomonas campestris pv. campestris were significantly more active than those from Agrobacterium tumefaciens, which must maintain host cell viability during infection. The structure of muropeptide components and the distinctive differences are described. Differing defense-eliciting abilities appear to depend on subtle structural differences in either carbohydrate or peptide groups.

  • The peculiar structural features of kiwi fruit pectin methylesterase: amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor.

    Publication Date: 01/04/2008 on Proteins
    by Ciardiello MA, D'Avino R, Amoresano A, Tuppo L, Carpentieri A, Carratore V, Tamburrini M, Giovane A, Pucci P, Camardella L
    DOI: 10.1002/prot.21681

    Pectin methylesterase (PME) from kiwi fruit (Actinidia deliciosa) is a glycoprotein, showing an apparent molecular mass of 50 kDa upon size exclusion chromatography and SDS-PAGE. The primary structure, elucidated by direct sequencing of the protein, comprises 321 amino acid residues providing a molecular mass of 35 kDa. The protein has an acetylated Thr residue at the amino terminus and five N-glycosylation consensus sequences, four of which are actually glycosylated. A careful investigation of the oligosaccharide structures demonstrated that PME glycans belong to complex type oligosaccharides essentially consisting of xylosylated polyfucosylated biantennary structures. Alignment with known mature plant PME sequences indicates that the postulated active site residues are conserved. Kiwi PME activity is inhibited following the interaction with the proteinaceous inhibitor PMEI, isolated from the same source. Gel-filtration experiments show that kiwi PME/PMEI complex is stable in a large pH range and dissociates only at pH 10.0. Modeling of the interaction with the inhibitor was performed by using the crystal structure of the complex between kiwi PMEI and tomato PME as a template. The model shows that the binding site is the same reported for tomato PME. However, additional salt link interactions are found to connect the external loops of kiwi PME to PMEI. This finding may explain the higher pH stability of the complex formed by the two kiwi proteins respect to that formed by PMEI and tomato PME.

  • A rapid and selective mass spectrometric method for the identification of nitrated proteins.

    Publication Date: 01/01/2008 on Methods in molecular biology (Clifton, N.J.)
    by Amoresano A, Chiappetta G, Pucci P, Marino G
    DOI: 10.1007/978-1-60327-517-0_2

    The nitration of protein tyrosine residues represents an important posttranslational modification during development, oxidative stress, and biological aging. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels, from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Identification of the specific targets of protein oxidation was previously accomplished using immunoprecipitation techniques followed by immunochemical detection. Here, we report a totally new approach involving dansyl chloride labeling of the nitration sites which relies on the enormous potential of MS(n) analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and a MS3 analysis. The novel labeling procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.

  • Hb Foggia or alpha 117(GH5)Phe -> Ser: a new alpha 2 globin allele affecting the alpha Hb-AHSP interaction.

    Publication Date: 01/01/2008 on Haematologica
    by Lacerra G, Scarano C, Musollino G, Flagiello A, Pucci P, Carestia C
    DOI: 10.3324/haematol.11789

    We report a novel alpha2-globin gene allele with the mutation cod 117 TTC>TCC or alpha 117(GH5)Phe>Ser detected in three carriers with alpha-thalassemia phenotype. The mutated mRNA was present in the reticulocytes in the same amount as the normal one, but no chain or hemoglobin variant were detected. Most likely the amino acid substitution impairs the interaction of the alpha-chain variant with the AHSP and prevents its stabilizing effect, thus leading to the alpha-chain pool reduction.

  • Functional proteomics: protein-protein interactions in vivo.

    Publication Date: 01/12/2007 on The Italian journal of biochemistry
    by Monti M, Cozzolino M, Cozzolino F, Tedesco R, Pucci P

    Functional proteomics constitutes an emerging research area in the proteomic field focused to two major targets, the elucidation of biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. Understanding protein functions as well as unravelling molecular mechanisms within the cell is then depending on the identification of the interacting protein partners. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism would in fact be strongly suggestive of its biological function. Furthermore, a detailed description of the cellular signalling pathways might greatly benefit from the elucidation of protein-protein interactions in the cell. Isolation of functional protein complexes essentially rely on affinity-based procedures. The protein of interest and its specific partners can be fished out from the cellular extract by using a suitable ligand as a bait taking advantage of the specific binding properties of the ligand molecule immobilised on agarose-sepharose supports. Alternative strategies essentially relying on immunoprecipitation techniques have been introduced to allow purification of protein complexes formed in vivo within the cell. The gene coding for the bait tagged with an epitope against which good antibodies exist (FLAG, HA, c-myc, etc.), is transfected into the appropriate cell line and expressed in the cognate host. The cell extracts are immunoprecipitated with anti-tag monoclonal antibodies using suitable experimental conditions to avoid dissociation of the complexes. In both cases, protein components specifically recognised by the bait and retained on the agarose beads can then be eluted and fractionated by SDS-PAGE. The protein bands detected on the gel are in situ enzymatically digested and the resulting peptide mixtures analysed by capillary LC-MS/MS techniques leading to the identification of the protein interactors.