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
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.
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
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.
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
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.
by Ciardiello MA, D'Avino R, Amoresano A, Tuppo L, Carpentieri A, Carratore V, Tamburrini M, Giovane A, Pucci P, Camardella L
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.
on Methods in molecular biology (Clifton, N.J.)
by Amoresano A, Chiappetta G, Pucci P, Marino G
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.
by Lacerra G, Scarano C, Musollino G, Flagiello A, Pucci P, Carestia C
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.
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.
on The EMBO journal
by Zito E, Buono M, Pepe S, Settembre C, Annunziata I, Surace EM, Dierks T, Monti M, Cozzolino M, Pucci P, Ballabio A, Cosma MP
Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.
on Analytical chemistry
by Amoresano A, Chiappetta G, Pucci P, D'Ischia M, Marino G
Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. 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. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn 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 an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.
on Protein science : a publication of the Protein Society
by Giorgetti S, Stoppini M, Tennent GA, Relini A, Marchese L, Raimondi S, Monti M, Marini S, Østergaard O, Heegaard NH, Pucci P, Esposito G, Merlini G, Bellotti V
The lysine 58 cleaved and truncated variant of beta(2)-microglobulin (DeltaK58-beta2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the beta2-microglobulin (beta2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure DeltaK58-beta2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of beta2m. Using this approach, the two known principal isoforms found in beta2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (DeltaN6-beta2m). In contrast, we found no evidence for the presence of DeltaK58-beta2m.