Latest PUBLICATIONS

  • Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells.
    Publication Date: 24/10/2008, on The Journal of biological chemistry
    by Lebreton S, Paladino S, Zurzolo C
    DOI: 10.1074/jbc.M803819200

    To determine the roles of cholesterol and the actin cytoskeleton in apical and basolateral protein organization and sorting, we have performed comprehensive confocal fluorescence recovery after photobleaching analyses of apical and basolateral and raft- and non-raft-associated proteins, both at the plasma membrane and in the Golgi apparatus of polarized MDCK cells. We show that at both the apical and basolateral plasma membrane domains, raft-associated proteins diffuse faster than non-raft-associated proteins and that, different from the latter, they become restricted upon depletion of cholesterol. Furthermore, only transmembrane apical proteins are restricted by the actin network. This indicates that cholesterol-dependent domains exist both at the apical and basolateral membranes of polarized cells and that the actin cytoskeleton has a predominant role in the organization of transmembrane proteins independent of their association with rafts at the apical membrane. In the Golgi apparatus apical proteins appear to be segregated from the basolateral ones in a compartment that is sensitive both to cholesterol depletion and actin rearrangements. Furthermore, consistent with the role of actin rearrangements in apical protein sorting, we found that apical proteins exhibit a differential sensitivity to actin depolymerization in the Golgi of polarized and nonpolarized cells.

  • 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.

  • Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation.
    Publication Date: 01/10/2008, on Journal of cellular and molecular medicine
    by Forte A, Finicelli M, De Luca P, Quarto C, Onorati F, Santè P, Renzulli A, Galderisi U, Berrino L, De Feo M, Rossi F, Cotrufo M, Cascino A, Cipollaro M
    DOI: 10.1111/j.1582-4934.2008.00212.x

    Vascular injury aimed at stenosis removal induces local reactions often leading to restenosis. The aim of this study was a concerted transcriptomic-proteomics analysis of molecular variations in a model of rat carotid arteriotomy, to dissect the molecular pathways triggered by vascular surgical injury and to identify new potential anti-restenosis targets. RNA and proteins extracted from inbred Wistar Kyoro (WKY) rat carotids harvested 4 hrs, 48 hrs and 7 days after arteriotomy were analysed by Affymetrix rat microarrays and by bidimensional electrophoresis followed by liquid chromatography and tandem mass spectrometry, using as reference the RNA and the proteins extracted from uninjured rat carotids. Results were classified according to their biological function, and the most significant Kyoro Encyclopedia of Genes and Genomes (KEGG) pathways were identified. A total of 1163 mRNAs were differentially regulated in arteriotomy-injured carotids 4 hrs, 48 hrs and 7 days after injury (P < 0.0001, fold-change > or =2), while 48 spots exhibited significant changes after carotid arteriotomy (P < 0.05, fold-change > or =2). Among them, 16 spots were successfully identified and resulted to correspond to a set of 19 proteins. mRNAs were mainly involved in signal transduction, oxidative stress/inflammation and remodelling, including many new potential targets for limitation of surgically induced (re)stenosis (e.g. Arginase I, Kruppel like factors). Proteome analysis confirmed and extended the microrarray data, revealing time-dependent post-translational modifications of Hsp27, haptoglobin and contrapsin-like protease inhibitor 6, and the differential expression of proteins mainly involved in contractility. Transcriptomic and proteomic methods revealed functional categories with different preferences, related to the experimental sensitivity and to mechanisms of regulation. The comparative analysis revealed correlation between transcriptional and translational expression for 47% of identified proteins. Exceptions from this correlation confirm the complementarities of these approaches.

  • 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.

  • RAGE recycles at the plasma membrane in S100B secretory vesicles and promotes Schwann cells morphological changes.
    Publication Date: 01/10/2008, on Journal of cellular physiology
    by Perrone L, Peluso G, Melone MA
    DOI: 10.1002/jcp.21474

    RAGE is a multiligand receptor of the immunoglobulin superfamily involved in regeneration of injured peripheral nerve and cell motility. RAGE is implicated in the development of various chronic diseases, such as neurodegenerative disorders, inflammatory responses, and diabetic complications. The correlation between RAGE endocytic trafficking and RAGE function is still uninvestigated. S100B is one of the ligands of RAGE. The molecular mechanisms responsible of S100B translocation in exocytic vesicles are still poorly investigated. In the present study we elucidate the role of RAGE endocytic trafficking in promoting S100B secretion in Schwann cells. Here we show that RAGE-induced secretion of S100B requires phosphorylated caveolin1-dependent endocytosis of RAGE. Endocytosis of RAGE in response to ligand binding promotes the fusion of endosomes with S100B-positive secretory vesicles. Src promotes the fusion of endosomes with S100B-secretory vesicles. Inhibition of src induces RAGE degradation. RAGE-mediated src activation induces cav1 phosphorylation and relocalization in the perinuclear compartment. RAGE signaling and recycling are required for S100-induced Schwann cells morphological changes and are inhibited by high-glucose, suggesting a possible link between diabetes and peripheral nerve injury. Indeed, high glucose inhibits RAGE-mediated src activation. Src inhibition blocks RAGE recycling, S100B secretion, and morphological changes. In summary, we identified a novel pathway of vesicular trafficking required for the amplification of RAGE signaling and cytoskeleton dynamics that is potentially involved in the regeneration of injured peripheral nerve.

  • Role of autophagy during methamphetamine neurotoxicity.
    Publication Date: 01/10/2008, on Annals of the New York Academy of Sciences
    by Pasquali L, Lazzeri G, Isidoro C, Ruggieri S, Paparelli A, Fornai F
    DOI: 10.1196/annals.1432.016

    Methamphetamine causes nigrostriatal denervation and striatal dopamine loss, while sparing nigral cell bodies. Nigral dopamine neurons feature autophagic vacuoles and cytoplasmic alpha-synuclein-, ubiquitin- and parkin-positive inclusion-like bodies. On that basis, autophagy was considered essential in methamphetamine-induced neurotoxicity, but its neurotoxic or protective role has never been addressed. Here we review the gap between the descriptive evidence on activation of autophagy and the lack of knowledge about its role during methamphetamine intoxication. Our preliminary findings rule out a detrimental role for autophagy; this represents the first step in understanding the consequence of activation of autophagy in methamphetamine toxicity.

  • Akt induces apoptosis in neuroblastoma cells expressing a C98X vasopressin mutant following autophagy suppression.
    Publication Date: 01/10/2008, on Journal of neuroendocrinology
    by Castino R, Thepparit C, Bellio N, Murphy D, Isidoro C
    DOI: 10.1111/j.1365-2826.2008.01769.x

    Mutations in the arginine vasopressin (AVP)-neurophysin II (NP-II) gene that affect the folding and transport of the prohormone result in loss of secretion of the anti-diuretic hormone AVP from pituitary nerve terminals and cause autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). One such mutation consists of the replacement of a Cys residue at position 98 with a stop codon (C98X) in the AVP precursor (corresponding to C67X in NP domain). In neuroblastoma cells over-expressing this truncated AVP precursor autophagy, a macromolecular degradation process, was shown to be essential for assuring cell survival. In the present study, we investigated the role of the Akt pro-survival signalling in the regulation of autophagy and of apoptosis linked with the handling of C98X AVP. Impairing autophagy-lysosomal sequestration or cathepsin D (CD)-mediated proteolysis triggered the activation of the intrinsic death pathway of apoptosis in C98X-expressing cells, but not in the wild-type -AVP-expressing cells. This was shown by the expression of a Vps34 dominant negative, which down-regulates the PI3k class III-dependent signalling needed for autophagosome (APH) formation, by genetic silencing as a result of RNA interference (RNAi) of Lamp2, a protein indispensable for the fusion of APHs with lysosomes, and by RNAi silencing of the lysosomal protease CD. Ectopic expression of either the wild-type or the mutated C98X AVP altered neither the expression nor the phosphorylation of the pro-survival signalling molecule Akt. Strikingly, the ectopic adenoviral-directed expression of a constitutively active Akt, instead of preserving cell survival, resulted in the suppression of autophagy, and precipitated Bax-mediated cell death. The present data demonstrate the need for autophagy-mediated degradation of mutated C98X peptides, which otherwise become toxic to the cell, and suggest that, in the presence of mis-folded proteins, the stimulation of the Akt signalling counteracts the beneficial effects of autophagy and precipitates cell death. It follows that growth factors impinging on the Akt pathway may have deleterious effect in neurones expressing mutant neuropeptides. This can provide an explanation for the late onset and progressive neuronal cell loss observed in hypothalamic magnocellular neurones of adFNDI patients.

  • Identification of a synaptosome-associated form of BAG3 protein.
    Publication Date: 01/10/2008, on Cell cycle (Georgetown, Tex.)
    by Bruno AP, Festa M, Dal Piaz F, Rosati A, Turco MC, Giuditta A, Marzullo L
    DOI: 10.4161/cc.7.19.6774

  • Alzheimer's disease and other dementing conditions.
    Publication Date: 01/10/2008, on Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology
    by Tedeschi G, Cirillo M, Tessitore A, Cirillo S
    DOI: 10.1007/s10072-008-1003-5

    Dementia is a common and growing problem, with 20% of those over 80 years of age suffering from this disorder. The prospect of more effective treatments has caused an increasing demand for a more accurate and earlier diagnosis of different dementia syndromes. Neuroimaging techniques may have an important role in the clinical evaluation of dementia for early diagnosis, differential diagnosis and may help in the prediction of conversion to dementia in individuals at a higher risk of developing the disorder. Moreover, new MRI techniques might not only further broaden our understanding of the pathophysiology but also accelerate treatment discovery. This review will focus on the use of conventional and non-conventional MRI techniques to investigate dementias.

  • Stability and cations coordination of DNA and RNA 14-mer G-quadruplexes: a multiscale computational approach.
    Publication Date: 25/09/2008, on The journal of physical chemistry. B
    by Pagano B, Mattia CA, Cavallo L, Uesugi S, Giancola C, Fraternali F
    DOI: 10.1021/jp804036j

    Molecular dynamics simulations have been used to study the differences between two DNA and RNA 14-mer quadruplexes of analogous sequences. Their structures present a completely different fold: DNA forms a bimolecular quadruplex containing antiparallel strands and diagonal loops; RNA forms an intrastrand parallel quadruplex containing a G-tetrad and an hexad, which dimerizes by hexad stacking. We used a multiscale computational approach combining classical Molecular dynamics simulations and density functional theory calculations to elucidate the difference in stability of the 2-folds and their ability in coordinating cations. The presence of 2'-OH groups in the RNA promotes the formation of a large number of intramolecular hydrogen bonds that account for the difference in fold and stability of the two 14-mers. We observe that the adenines in the RNA quadruplex play a key role in conserving the geometry of the hexad. We predict the cation coordination mode of the two quadruplexes, not yet observed experimentally, and we offer a rationale for the corresponding binding energies involved.

  • In inclusion-body myositis muscle fibers Parkinson-associated DJ-1 is increased and oxidized.
    Publication Date: 15/09/2008, on Free radical biology & medicine
    by Terracciano C, Nogalska A, Engel WK, Wojcik S, Askanas V
    DOI: 10.1016/j.freeradbiomed.2008.05.030

    Sporadic inclusion-body myositis (s-IBM) is the most common muscle disease of older persons. The muscle-fiber molecular phenotype exhibits similarities to both Alzheimer-disease (AD) and Parkinson-disease (PD) brains, including accumulations of amyloid-beta, phosphorylated tau, alpha-synuclein, and parkin, as well as evidence of oxidative stress and mitochondrial abnormalities. Early-onset autosomal-recessive PD can be caused by mutations in the DJ-1 gene, leading to its inactivation. DJ-1 has antioxidative and mitochondrial-protective properties. In AD and PD brains, DJ-1 is increased and oxidized. We studied DJ-1 in 17 s-IBM and 18 disease-control and normal muscle biopsies by: (1) immunoblots of muscle homogenates and mitochondrial fractions; (2) real-time PCR; (3) oxyblots evaluating DJ-1 oxidation; (4) light- and electron-microscopic immunocytochemistry. Compared to controls, in s-IBM muscle fibers DJ-1 was: (a) increased in the soluble fraction, monomer 2-fold (P = 0.01), and dimer 2.8-fold (P = 0.004); (b) increased in the mitochondrial fraction; (c) highly oxidized; and (d) aggregated in about 15% of the abnormal muscle fibers. DJ-1 mRNA was increased 3.5-fold (P = 0.034). Accordingly, DJ-1 might play a role in human muscle disease, and thus not be limited to human CNS degenerations. In s-IBM muscle fibers, DJ-1 could be protecting these fibers against oxidative stress, including protection of mitochondria.

  • 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.

  • Left hand tactile agnosia after posterior callosal lesion.
    Publication Date: 01/09/2008, on Cortex; a journal devoted to the study of the nervous system and behavior
    by Balsamo M, Trojano L, Giamundo A, Grossi D
    DOI: 10.1016/j.cortex.2008.01.003

    We report a patient with a hemorrhagic lesion encroaching upon the posterior third of the corpus callosum but sparing the splenium. She showed marked difficulties in recognizing objects and shapes perceived through her left hand, while she could appreciate elementary sensorial features of items tactually presented to the same hand flawlessly. This picture, corresponding to classical descriptions of unilateral associative tactile agnosia, was associated with finger agnosia of the left hand. This very unusual case report can be interpreted as an instance of disconnection syndrome, and allows a discussion of mechanisms involved in tactile object recognition.

  • Selective motor imagery defect in patients with locked-in syndrome.
    Publication Date: 01/09/2008, on Neuropsychologia
    by Conson M, Sacco S, Sarà M, Pistoia F, Grossi D, Trojano L
    DOI: 10.1016/j.neuropsychologia.2008.04.015

    Recent studies indicate that motor imagery is subserved by activation of motor information. However, at present it is not clear whether the sparing of motor efferent pathways is necessary to perform a motor imagery task. To clarify this issue, we required patients with a selective, severe de-efferentation (locked-in syndrome, LIS) to mentally manipulate hands and three-dimensional objects. Compared with normal controls, LIS patients showed a profound impairment on a modified version of the hand-laterality task and a normal performance on mental rotation of abstract items. Moreover, LIS patients did not present visuomotor compatibility effects between anatomical side of hands and spatial location of stimuli on the computer screen. Such findings confirmed that the motor system is involved in mental simulation of action but not in mental manipulation of visual images. To explain LIS patients' inability in manipulating hand representations, we suggested that the pontine lesion, both determined a complete de-efferentation, and affected a component of the motor system, which is crucial for mental representation of body parts, probably the neural connections between parietal lobes and cerebellum.

  • Therapeutic targeting of the stem cell niche in experimental hindlimb ischemia.
    Publication Date: 01/09/2008, on Nature clinical practice. Cardiovascular medicine
    by Napoli C, William-Ignarro S, Byrns R, Balestrieri ML, Crimi E, Farzati B, Mancini FP, de Nigris F, Matarazzo A, D'Amora M, Abbondanza C, Fiorito C, Giovane A, Florio A, Varricchio E, Palagiano A, Minucci PB, Tecce MF, Giordano A, Pavan A, Ignarro LJ
    DOI: 10.1038/ncpcardio1214

    The custom microenvironment 'vascular niche' is a potential therapeutic target for several pathophysiological conditions. Osteoblasts regulate the hematopoietic stem cell niche, and activation of the parathyroid hormone (PTH) receptor can increase the number of cells mobilized into the bloodstream.