on Critical reviews in biochemistry and molecular biology
by Lebreton S, Zurzolo C, Paladino S
Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. The presence of both glycolipid anchor and protein portion confers them unique features. GPI-APs are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, neuritogenesis, and immune response. Likewise other plasma membrane proteins, the spatio-temporal organization of GPI-APs is critical for their biological activities in physiological conditions. In this review, we will summarize the latest findings on plasma membrane organization of GPI-APs and the mechanism of its regulation in different cell types. We will also examine the involvement of specific GPI-APs namely the prion protein PrP, the Folate Receptor alpha and the urokinase plasminogen activator receptor in human diseases focusing on neurodegenerative diseases and cancer.
on Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
by Paladino S, Conte A, Caggiano R, Pierantoni GM, Faraonio R
A general hallmark of neurological diseases is the loss of redox homeostasis that triggers oxidative damages to biomolecules compromising neuronal function. Under physiological conditions the steady-state concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are finely regulated for proper cellular functions. Reduced surveillance of endogenous antioxidant defenses and/or increased ROS/RNS production leads to oxidative stress with consequent alteration of physiological processes. Neuronal cells are particularly susceptible to ROS/RNS due to their biochemical composition. Overwhelming evidences indicate that nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-linked pathways are involved in protective mechanisms against oxidative stress by regulating antioxidant and phase II detoxifying genes. As such, Nrf2 deregulation has been linked to both aging and pathogenesis of many human chronic diseases, including neurodegenerative ones such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Nrf2 activity is tightly regulated by a fine balance between positive and negative modulators. A better understanding of the regulatory mechanisms underlying Nrf2 activity could help to develop novel therapeutic interventions to prevent, slow down or possibly reverse various pathological states. To this end, microRNAs (miRs) are attractive candidates because they are linked to intracellular redox status being regulated and, post-transcriptionally, regulating key components of ROS/RNS pathways, including Nrf2.
on Molecular therapy. Methods & clinical development
by De Pasquale V, Sarogni P, Pistorio V, Cerulo G, Paladino S, Pavone LM
Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by the deficiency of lysosomal enzymes needed to catabolize glycosaminoglycans (GAGs). Four therapeutic options are currently considered: enzyme replacement therapy, substrate reduction therapy, gene therapy, and hematopoietic stem cell transplantation. However, while some of them exhibit limited clinical efficacy and require high costs, others are still in development. Therefore, alternative treatments for MPSs need to be explored. Here we describe an innovative therapeutic approach based on the use of a recombinant protein that is able to bind the excess of extracellular accumulated heparan sulfate (HS). We demonstrate that this protein is able to reduce lysosomal defects in primary fibroblasts from MPS I and MPS IIIB patients. We also show that, by masking the excess of extracellular accumulated HS in MPS fibroblasts, fibroblast growth factor (FGF) signal transduction can be positively modulated. We, therefore, suggest the use of a competitive binding molecule for HS in MPSs as an alternative strategy to prevent the detrimental extracellular substrate storage.
on Cell death & disease
by Fasano D, Parisi S, Pierantoni GM, De Rosa A, Picillo M, Amodio G, Pellecchia MT, Barone P, Moltedo O, Bonifati V, De Michele G, Nitsch L, Remondelli P, Criscuolo C, Paladino S
Recently, a new form of autosomal recessive early-onset parkinsonism (PARK20), due to mutations in the gene encoding the phosphoinositide phosphatase, Synaptojanin 1 (Synj1), has been reported. Several genes responsible for hereditary forms of Parkinson's disease are implicated in distinct steps of the endolysosomal pathway. However, the nature and the degree of endocytic membrane trafficking impairment in early-onset parkinsonism remains elusive. Here, we show that depletion of Synj1 causes drastic alterations of early endosomes, which become enlarged and more numerous, while it does not affect the morphology of late endosomes both in non-neuronal and neuronal cells. Moreover, Synj1 loss impairs the recycling of transferrin, while it does not alter the trafficking of the epidermal growth factor receptor. The ectopic expression of Synj1 restores the functions of early endosomes, and rescues these trafficking defects in depleted cells. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts of PARK20 patients. Our data indicate that Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types, and highlight defective cellular pathways in PARK20. In addition, they strengthen the link between endosomal trafficking and Parkinson's disease.
on Pharmaceutical research
by Melone MAB, Dato C, Paladino S, Coppola C, Trebini C, Giordana MT, Perrone L
Oxidative stress is a hallmark of Alzheimer's Disease (AD) and promotes tau phosphorylation. Since Thioredoxin Interacting protein (TXNIP), the inhibitor of the anti-oxidant system of Thioredoxin, is up regulated in the hippocampus of AD patients, we investigated whether TXNIP plays a role in promoting tau phosphorylation and whether Verapamil, an inhibitor of TXNIP expression, prevents TXNIP downstream effects.
on Cell death & disease
by De Pasquale V, Pezone A, Sarogni P, Tramontano A, Schiattarella GG, Avvedimento VE, Paladino S, Pavone LM
Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease caused by the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The defective lysosomal clearance of undigested HS results in dysfunction of multiple tissues and organs. We recently demonstrated that the murine model of MPS IIIB develops cardiac disease, valvular abnormalities, and ultimately heart failure. To address the molecular mechanisms governing cardiac dysfunctions in MPS IIIB, we generated a model of the disease by silencing NAGLU gene expression in H9C2 rat cardiomyoblasts. NAGLU-depleted H9C2 exhibited accumulation of abnormal lysosomes and a hypertrophic phenotype. Furthermore, we found the specific activation of the epidermal growth factor receptor (EGFR), and increased phosphorylation levels of extracellular signal-regulated kinases (ERKs) in NAGLU-depleted H9C2. The inhibition of either EGFR or ERKs, using the selective inhibitors AG1478 and PD98059, resulted in the reduction of both lysosomal aberration and hypertrophy in NAGLU-depleted H9C2. We also found increased phosphorylation of c-Src and a reduction of the hypertrophic response in NAGLU-depleted H9C2 transfected with a dominant-negative c-Src. However, c-Src phosphorylation remained unaffected by AG1478 treatment, posing c-Src upstream EGFR activation. Finally, heparin-binding EGF-like growth factor (HB-EGF) protein was found overexpressed in our MPS IIIB cellular model, and its silencing reduced the hypertrophic response. These results indicate that both c-Src and HB-EGF contribute to the hypertrophic phenotype of NAGLU-depleted cardiomyoblasts by synergistically activating EGFR and subsequent signaling, thus suggesting that EGFR pathway inhibition could represent an effective therapeutic approach for MPS IIIB cardiac disease.
on The Biochemical journal
by Paladino S, Lebreton S, Lelek M, Riccio P, De Nicola S, Zimmer C, Zurzolo C
Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model.
on Journal of the National Cancer Institute
by Paladino S, Melillo RM
on Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology
by Cardinale A, Fusco FR, Paldino E, Giampà C, Marino M, Nuzzo MT, D'Angelo V, Laurenti D, Straccia G, Fasano D, Sarnataro D, Squillaro T, Paladino S, Melone MAB
Neuroglobin (Ngb) is expressed in the central and peripheral nervous system, cerebrospinal fluid, retina, and endocrine tissues where it is involved in binding O2 and other gasotransmitters. Several studies have highlighted its endogenous neuroprotective function. Huntington's disease (HD), a dominant hereditary disease, is characterized by the gradual loss of neurons in discrete areas of the central nervous system. We analyzed the expression of Ngb in the brain tissue of a mouse model of HD, in order to define the role of Ngb with respect to individual cell type vulnerability in HD and to gender and age of mice. Our results showed different expressions of Ngb among neurons of a specific region and between different brain regions. We evidenced a decreased intensity of Ngb at 13 weeks of age, compared to 7 weeks of age. The double immunofluorescence and fluorescence resonance energy transfer (FRET) experiments showed that the co-localization between Ngb and huntingtin at the subcellular level was not close enough to account for a direct interaction. We also observed a different expression of Ngb in the striatum, depending on the sex and age of animals. These findings provide the first experimental evidence for an adaptive response of Ngb in HD, suggesting that Ngb may exert neuroprotective effects in HD beyond its role in reducing sensitivity to oxidative stress.
on ACS applied materials & interfaces
by Silvestri B, Vitiello G, Luciani G, Calcagno V, Costantini A, Gallo M, Parisi S, Paladino S, Iacomino M, D'Errico G, Caso MF, Pezzella A, d'Ischia M
We disclose herein the first example of stable monodispersed hybrid nanoparticles (termed MelaSil-NPs) made up of eumelanin biopolymer intimately integrated into a silica nanoscaffold matrix and endowed with high antioxidant and cytoprotective effects associated with a specific subcellular localization. MelaSil-NPs have been fabricated by an optimized sol-gel methodology involving ammonia-induced oxidative polymerization of a covalent conjugate of the eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA) with 3-aminopropyltriethoxysilanes (APTS). They displayed a round-shaped (ca. 50-80 nm) morphology, exhibited the typical electron paramagnetic resonance signal of eumelanin biopolymers, and proved effective in promoting decomposition of hydrogen peroxide under physiologically relevant conditions. When administered to human ovarian cancer cells (A2780) or cervical cancer cells (HeLa), MelaSil-NPs were rapidly internalized and colocalized with lysosomes and exerted efficient protecting effects against hydrogen peroxide-induced oxidative stress and cytotoxicity.