Publications by authors named "Daniela Sarnataro"

48 Publications

Nuclear FGFR2 Interacts with the MLL-AF4 Oncogenic Chimera and Positively Regulates Gene Expression in t(4;11) Leukemia Cells.

Int J Mol Sci 2021 Apr 28;22(9). Epub 2021 Apr 28.

CEINGE Advanced Biotechnologies s.c. a r.l., via G. Salvatore, 486, 80145 Naples, Italy.

The chromosomal translocation t(4;11) marks an infant acute lymphoblastic leukemia associated with dismal prognosis. This rearrangement leads to the synthesis of the MLL-AF4 chimera, which exerts its oncogenic activity by upregulating transcription of genes involved in hematopoietic differentiation. Crucial for chimera's aberrant activity is the recruitment of the AF4/ENL/P-TEFb protein complex. Interestingly, a molecular interactor of AF4 is fibroblast growth factor receptor 2 (FGFR2). We herein analyze the role of FGFR2 in the context of leukemia using t(4;11) leukemia cell lines. We revealed the interaction between MLL-AF4 and FGFR2 by immunoprecipitation, western blot, and immunofluorescence experiments; we also tested the effects of FGFR2 knockdown, FGFR2 inhibition, and FGFR2 stimulation on the expression of the main MLL-AF4 target genes, i.e., and . Our results show that FGFR2 and MLL-AF4 interact in the nucleus of leukemia cells and that FGFR2 knockdown, which is associated with decreased expression of and , impairs the binding of MLL-AF4 to the promoter. We also show that stimulation of leukemia cells with FGF2 increases nuclear level of FGFR2 in its phosphorylated form, as well as and expression. In contrast, preincubation with the ATP-mimetic inhibitor PD173074, before FGF2 stimulation, reduced FGFR2 nuclear amount and and transcript level, thereby indicating that MLL-AF4 aberrant activity depends on the nuclear availability of FGFR2. Overall, our study identifies FGFR2 as a new and promising therapeutic target in t(4;11) leukemia.
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http://dx.doi.org/10.3390/ijms22094623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124917PMC
April 2021

Probiotics, prebiotics and their role in Alzheimer's disease.

Neural Regen Res 2021 Sep;16(9):1768-1769

Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.

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http://dx.doi.org/10.4103/1673-5374.306072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8328749PMC
September 2021

Inhibition of 37/67kDa Laminin-1 Receptor Restores APP Maturation and Reduces Amyloid-β in Human Skin Fibroblasts from Familial Alzheimer's Disease.

J Pers Med 2020 Nov 16;10(4). Epub 2020 Nov 16.

Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II", Via S. Pansini 5, 80131 Naples, Italy.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder caused by protein misfolding and aggregation, affecting brain function and causing dementia. Amyloid beta (Aβ), a peptide deriving from amyloid precursor protein (APP) cleavage by-and γ-secretases, is considered a pathological hallmark of AD. Our previous study, together with several lines of evidence, identified a strict link between APP, Aβ and 37/67kDa laminin receptor (LR), finding the possibility to regulate intracellular APP localization and maturation through modulation of the receptor. Here, we report that in fibroblasts from familial AD (fAD), APP was prevalently expressed as an immature isoform and accumulated preferentially in the transferrin-positive recycling compartment rather than in the Golgi apparatus. Moreover, besides the altered mitochondrial network exhibited by fAD patient cells, the levels of pAkt and pGSK3 were reduced in respect to healthy control fibroblasts and were accompanied by an increased amount of secreted Aβ in conditioned medium from cell cultures. Interestingly, these features were reversed by inhibition of 37/67kDa LR by NSC47924 a small molecule that was able to rescue the "typical" APP localization in the Golgi apparatus, with consequences on the Aβ level and mitochondrial network. Altogether, these findings suggest that 37/67kDa LR modulation may represent a useful tool to control APP trafficking and Aβ levels with implications in Alzheimer's disease.
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http://dx.doi.org/10.3390/jpm10040232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712490PMC
November 2020

Liposome-Embedding Silicon Microparticle for Oxaliplatin Delivery in Tumor Chemotherapy.

Pharmaceutics 2020 Jun 17;12(6). Epub 2020 Jun 17.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli "Federico II", 80131 Napoli, Italy.

Mesoporous silicon microparticles (MSMPs) can incorporate drug-carrying nanoparticles (NPs) into their pores. An NP-loaded MSMP is a multistage vector (MSV) that forms a Matryoshka-like structure that protects the therapeutic cargo from degradation and prevents its dilution in the circulation during delivery to tumor cells. We developed an MSV constituted by 1 µm discoidal MSMPs embedded with PEGylated liposomes containing oxaliplatin (oxa) which is a therapeutic agent for colorectal cancer (CRC). To obtain extra-small liposomes able to fit the 60 nm pores of MSMP, we tested several liposomal formulations, and identified two optimal compositions, with a prevalence of the rigid lipid 1,2-distearoyl-sn-glycero-3-phosphocholine and of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. To improve the MSV assembly, we optimized the liposome-loading inside the MSMP and achieved a five-fold increase of the payload using an innovative lyophilization approach. This procedure also increased the load and limited dimensional changes of the liposomes released from the MSV in vitro. Lastly, we found that the cytotoxic efficacy of oxa-loaded liposomes and-oxa-liposome-MSV in CRC cell culture was similar to that of free oxa. This study increases knowledge about extra-small liposomes and their loading into porous materials and provides useful hints about alternative strategies for designing drug-encapsulating NPs.
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http://dx.doi.org/10.3390/pharmaceutics12060559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355455PMC
June 2020

ZSCAN4 mouse embryonic stem cells have an oxidative and flexible metabolic profile.

EMBO Rep 2020 06 18;21(6):e48942. Epub 2020 May 18.

Department of Biology, University of Naples Federico II, Naples, Italy.

Cultured mouse embryonic stem cells are a heterogeneous population with diverse differentiation potential. In particular, the subpopulation marked by Zscan4 expression has high stem cell potency and shares with 2 cell stage preimplantation embryos both genetic and epigenetic mechanisms that orchestrate zygotic genome activation. Although embryonic de novo genome activation is known to rely on metabolites, a more extensive metabolic characterization is missing. Here we analyze the Zscan4 mouse stem cell metabolic phenotype associated with pluripotency maintenance and cell reprogramming. We show that Zscan4 cells have an oxidative and adaptable metabolism, which, on one hand, fuels a high bioenergetic demand and, on the other hand, provides intermediate metabolites for epigenetic reprogramming. Our findings enhance our understanding of the metastable Zscan4 stem cell state with potential applications in regenerative medicine.
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http://dx.doi.org/10.15252/embr.201948942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271321PMC
June 2020

New Insights into the Molecular Bases of Familial Alzheimer's Disease.

J Pers Med 2020 Apr 19;10(2). Epub 2020 Apr 19.

Department of Molecular Medicine and Medical Biotechnology, Federico II University, via S. Pansini 5-80131 Naples, Italy.

Like several neurodegenerative disorders, such as Prion and Parkinson diseases, Alzheimer's disease (AD) is characterized by spreading mechanism of aggregated proteins in the brain in a typical "prion-like" manner. Recent genetic studies have identified in four genes associated with inherited AD (amyloid precursor protein-, Presenilin-1, Presenilin-2 and Apolipoprotein E), rare mutations which cause dysregulation of APP processing and alterations of folding of the derived amyloid beta peptide (A). Accumulation and aggregation of A in the brain can trigger a series of intracellular events, including hyperphosphorylation of tau protein, leading to the pathological features of AD. However, mutations in these four genes account for a small of the total genetic risk for familial AD (FAD). Genome-wide association studies have recently led to the identification of additional AD candidate genes. Here, we review an update of well-established, highly penetrant FAD-causing genes with correlation to the protein misfolding pathway, and novel emerging candidate FAD genes, as well as inherited risk factors. Knowledge of these genes and of their correlated biochemical cascade will provide several potential targets for treatment of AD and aging-related disorders.
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http://dx.doi.org/10.3390/jpm10020026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354425PMC
April 2020

APP Maturation and Intracellular Localization Are Controlled by a Specific Inhibitor of 37/67 kDa Laminin-1 Receptor in Neuronal Cells.

Int J Mol Sci 2020 Mar 4;21(5). Epub 2020 Mar 4.

Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II", Via S. Pansini 5, 80131 Naples, Italy.

Amyloid precursor protein (APP) is processed along both the nonamyloidogenic pathway preventing amyloid beta peptide (Aβ) production and the amyloidogenic pathway, generating Aβ, whose accumulation characterizes Alzheimer's disease. Items of evidence report that the intracellular trafficking plays a key role in the generation of Aβ and that the 37/67 kDa LR (laminin receptor), acting as a receptor for Aβ, may mediate Aβ-pathogenicity. Moreover, findings indicating interaction between the receptor and the key enzymes involved in the amyloidogenic pathway suggest a strong link between 37/67 kDa LR and APP processing. We show herein that the specific 37/67 kDa LR inhibitor, NSC48478, is able to reversibly affect the maturation of APP in a pH-dependent manner, resulting in the partial accumulation of the immature APP isoforms (unglycosylated/acetylated forms) in the endoplasmic reticulum (ER) and in transferrin-positive recycling endosomes, indicating alteration of the APP intracellular trafficking. These effects reveal NSC48478 inhibitor as a novel small molecule to be tested in disease conditions, mediated by the 37/67 kDa LR and accompanied by inactivation of ERK1/2 (extracellular signal-regulated kinases) signalling and activation of Akt (serine/threonine protein kinase) with consequent inhibition of GSK3β.
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http://dx.doi.org/10.3390/ijms21051738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084285PMC
March 2020

Microbiome Influence in the Pathogenesis of Prion and Alzheimer's Diseases.

Int J Mol Sci 2019 Sep 23;20(19). Epub 2019 Sep 23.

Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, via Pansini 5, 80131 Naples, Italy.

Misfolded and abnormal β-sheets forms of wild-type proteins, such as cellular prion protein (PrP) and amyloid beta (Aβ), are believed to be the vectors of neurodegenerative diseases, prion and Alzheimer's disease (AD), respectively. Increasing evidence highlights the "prion-like" seeding of protein aggregates as a mechanism for pathological spread in AD, tauopathy, as well as in other neurodegenerative diseases, such as Parkinson's. Mutations in both PrP and Aβ precursor protein (APP), have been associated with the pathogenesis of these fatal disorders with clear evidence for their pathogenic significance. In addition, a critical role for the gut microbiota is emerging; indeed, as a consequence of gut-brain axis alterations, the gut microbiota has been involved in the regulation of Aβ production in AD and, through the microglial inflammation, in the amyloid fibril formation, in prion diseases. Here, we aim to review the role of microbiome ("the other human genome") alterations in AD and prion disease pathogenesis.
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http://dx.doi.org/10.3390/ijms20194704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801937PMC
September 2019

Crosstalk between 14-3-3θ and AF4 enhances MLL-AF4 activity and promotes leukemia cell proliferation.

Cell Oncol (Dordr) 2019 Dec 6;42(6):829-845. Epub 2019 Sep 6.

IRCCS SDN, 80143, Naples, Italy.

Purpose: The t(4;11)(q21;q23) translocation characterizes a form of acute lymphoblastic leukemia with a poor prognosis. It results in a fusion gene encoding a chimeric transcription factor, MLL-AF4, that deregulates gene expression through a variety of still controversial mechanisms. To provide new insights into these mechanisms, we examined the interaction between AF4, the most common MLL fusion partner, and the scaffold protein 14-3-3θ, in the context of t(4;11)-positive leukemia.

Methods: Protein-protein interactions were analyzed using immunoprecipitation and in vitro binding assays, and by fluorescence microscopy in t(4;11)-positive RS4;11 and MV4-11 leukemia cells and in HEK293 cells. Protein and mRNA expression levels were determined by Western blotting and RT-qPCR, respectively. A 5-bromo-2'-deoxyuridine assay and an annexin V/propidium iodide assay were used to assess proliferation and apoptosis rates, respectively, in t(4;11)-positive and control cells. Chromatin immunoprecipitation was performed to assess binding of 14-3-3θ and AF4 to a specific promoter element.

Results: We found that AF4 and 14-3-3θ are nuclear interactors, that 14-3-3θ binds Ser of AF4 and that 14-3-3θ forms a complex with MLL-AF4. In addition, we found that in t(4;11)-positive cells, 14-3-3θ knockdown decreased the expression of MLL-AF4 target genes, induced apoptosis and hampered cell proliferation. Moreover, we found that 14-3-3θ knockdown impaired the recruitment of AF4, but not of MLL-AF4, to target chromatin. Overall, our data indicate that the activity of the chimeric transcription factor MLL-AF4 depends on the cellular availability of 14-3-3θ, which triggers the transactivating function and subsequent degradation of AF4.

Conclusions: From our data we conclude that the scaffold protein 14-3-3θ enhances the aberrant activity of the chimeric transcription factor MLL-AF4 and, therefore, represents a new player in the molecular pathogenesis of t(4;11)-positive leukemia and a new promising therapeutic target.
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http://dx.doi.org/10.1007/s13402-019-00468-6DOI Listing
December 2019

HMGA1 negatively regulates NUMB expression at transcriptional and post transcriptional level in glioblastoma stem cells.

Cell Cycle 2019 07 22;18(13):1446-1457. Epub 2019 May 22.

a Istituto di Endocrinologia ed Oncologia Sperimentale - CNR c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche , Università degli Studi di Napoli "Federico II" , Naples , Italy.

Glioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2-3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth. We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division. Since NUMB expression is fundamental for the fulfillment of asymmetric division in stem cells, and is lost or reduced in many tumors, including GBM, we have investigated the ability of HMGA1 to regulate NUMB expression. Here, we show that HMGA1 negatively regulates NUMB expression at transcriptional level, by binding its promoter and counteracting c/EBP-β and at posttranscriptional level, by regulating the expression of MSI1 and of miR-146a. Finally, we report that HMGA1 knockdown-induced NUMB upregulation leads to the downregulation of the NOTCH1 pathway. Therefore, the data reported here indicate that HMGA1 negatively regulates NUMB expression in BTSCs, further supporting HMGA1 targeting as innovative and effective anti-cancer therapy.
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http://dx.doi.org/10.1080/15384101.2019.1618541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592240PMC
July 2019

Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system.

J Cell Biol 2019 03 18;218(3):1055-1065. Epub 2019 Jan 18.

Telethon Institute of Genetics and Medicine, Pozzuoli, Italy

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.
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http://dx.doi.org/10.1083/jcb.201812020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400564PMC
March 2019

Attempt to Untangle the Prion-Like Misfolding Mechanism for Neurodegenerative Diseases.

Int J Mol Sci 2018 Oct 9;19(10). Epub 2018 Oct 9.

Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, School of Medicine, Via S. Pansini 5, 80131 Naples, Italy.

The misfolding and aggregation of proteins is the neuropathological hallmark for numerous diseases including Alzheimer's disease, Parkinson's disease, and prion diseases. It is believed that misfolded and abnormal β-sheets forms of wild-type proteins are the vectors of these diseases by acting as seeds for the aggregation of endogenous proteins. Cellular prion protein (PrP) is a glycosyl-phosphatidyl-inositol (GPI) anchored glycoprotein that is able to misfold to a pathogenic isoform PrP, the causative agent of prion diseases which present as sporadic, dominantly inherited and transmissible infectious disorders. Increasing evidence highlights the importance of prion-like seeding as a mechanism for pathological spread in Alzheimer's disease and Tauopathy, as well as other neurodegenerative disorders. Here, we report the latest findings on the mechanisms controlling protein folding, focusing on the ER (Endoplasmic Reticulum) quality control of GPI-anchored proteins and describe the "prion-like" properties of amyloid-β and tau assemblies. Furthermore, we highlight the importance of pathogenic assemblies interaction with protein and lipid membrane components and their implications in both prion and Alzheimer's diseases.
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http://dx.doi.org/10.3390/ijms19103081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213118PMC
October 2018

Protein Syndesmos is a novel RNA-binding protein that regulates primary cilia formation.

Nucleic Acids Res 2018 12;46(22):12067-12086

Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Napoli, Italy.

Syndesmos (SDOS) is a functionally poorly characterized protein that directly interacts with p53 binding protein 1 (53BP1) and regulates its recruitment to chromatin. We show here that SDOS interacts with another important cancer-linked protein, the chaperone TRAP1, associates with actively translating polyribosomes and represses translation. Moreover, we demonstrate that SDOS directly binds RNA in living cells. Combining individual gene expression profiling, nucleotide crosslinking and immunoprecipitation (iCLIP), and ribosome profiling, we discover several crucial pathways regulated post-transcriptionally by SDOS. Among them, we identify a small subset of mRNAs responsible for the biogenesis of primary cilium that have been linked to developmental and degenerative diseases, known as ciliopathies, and cancer. We discover that SDOS binds and regulates the translation of several of these mRNAs, controlling cilia development.
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http://dx.doi.org/10.1093/nar/gky873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294507PMC
December 2018

An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population.

Int J Mol Sci 2018 06 27;19(7). Epub 2018 Jun 27.

Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, via S. Pansini 5, 80131 Naples, Italy.

The H1069Q substitution is the most frequent mutation of the Cu transporter ATP7B that causes Wilson disease in the Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes, but, in the presence of excessive Cu, it relocates to the endo-lysosomal compartment to excrete Cu via bile canaliculi. In contrast, ATP7B-H1069Q is strongly retained in the ER, does not reach the Golgi complex and fails to move to the endo-lysosomal compartment in the presence of excessive Cu, thus causing toxic Cu accumulation. We have previously shown that, in transfected cells, the small heat-shock protein αB-crystallin is able to correct the mislocalization of ATP7B-H1069Q and its trafficking in the presence of Cu overload. Here, we first show that the α-crystallin domain of αB-crystallin mimics the effect of the full-length protein, whereas the N- and C-terminal domains have no such effect. Next, and most importantly, we demonstrate that a twenty-residue peptide derived from the α-crystallin domain of αB-crystallin fully rescues Golgi localization and the trafficking response of ATP7B-H1069Q in the presence of Cu overload. In addition, we show that this peptide interacts with the mutant transporter in the live cell. These results open the way to attempt developing a pharmacologically active peptide to specifically contrast the Wilson disease form caused by the ATP7B-H1069Q mutant.
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http://dx.doi.org/10.3390/ijms19071892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073935PMC
June 2018

Multimodal imaging for a theranostic approach in a murine model of B-cell lymphoma with engineered nanoparticles.

Nanomedicine 2018 02 22;14(2):483-491. Epub 2017 Nov 22.

Dipartimento di Scienze Biomediche Avanzate, Università degli Studi di Napoli Federico II, Napoli, Italia; IBB, CNR, Napoli, Italia; CEINGE Biotecnologie Avanzate, s.c.ar.l., Napoli, Italia. Electronic address:

Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.
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http://dx.doi.org/10.1016/j.nano.2017.11.016DOI Listing
February 2018

Cell Biology of Prion Protein.

Prog Mol Biol Transl Sci 2017 29;150:57-82. Epub 2017 Jul 29.

University of Naples "Federico II", Naples, Italy; Unité de Trafic Membranaire et Pathogenese, Institut Pasteur, Paris, France.

Cellular prion protein (PrP) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The precise function of PrP remains elusive but may depend upon its cellular localization. PrP misfolds to a pathogenic isoform PrP, the causative agent of neurodegenerative prion diseases. Nonetheless some forms of prion disease develop in the apparent absence of infectious PrP, suggesting that molecular species of PrP distinct from PrP may represent the primary neurotoxic culprits. Indeed, in some inherited cases of human prion disease, the predominant form of PrP detectable in the brain is not PrP but rather PrP, a transmembrane form of the protein. The relationship between the neurodegeneration occurring in prion diseases involving PrP and that associated with PrP remains unclear. However, the different membrane topology of the PrP mutants, as well as the presence of the GPI anchor, could influence both the function and the intracellular localization and trafficking of the protein, all being potentially very important in the pathophysiological mechanism that ultimately causes the disease. Here, we review the latest findings on the fundamental aspects of prions biology, from the PrP biosynthesis, function, and structure up to its intracellular traffic and analyze the possible roles of the different topological isoforms of the protein, as well as the GPI anchor, in the pathogenesis of the disease.
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http://dx.doi.org/10.1016/bs.pmbts.2017.06.018DOI Listing
May 2018

Regulation of sub-compartmental targeting and folding properties of the Prion-like protein Shadoo.

Sci Rep 2017 06 16;7(1):3731. Epub 2017 Jun 16.

Department of Molecular Medicine and Medical Biotechnology, University of Naples "Federico II", Via Pansini 5-80131, Naples, Italy.

Shadoo (Sho), a member of prion protein family, has been shown to prevent embryonic lethality in Prnp mice and to be reduced in the brains of rodents with terminal prion diseases. Sho can also affect PrP structural dynamics and can increase the prion conversion into its misfolded isoform (PrP), which is amyloidogenic and strictly related to expression, intracellular localization and association of PrP to lipid rafts. We reasoned that if Sho possesses a natural tendency to convert to amyloid-like forms in vitro, it should be able to exhibit "prion-like" properties, such as PK-resistance and aggregation state, also in live cells. We tested this hypothesis, by different approaches in neuronal cells, finding that Sho shows folding properties partially dependent on lipid rafts integrity whose alteration, as well as proteasomal block, regulated generation of intermediate Sho isoforms and exacerbated its misfolding. Moreover, a 18 kDa isoform of Sho, likely bearing the signal peptide, was targeted to mitochondria by interacting with the molecular chaperone TRAP1 which, in turn controlled Sho dual targeting to ER or mitochondria. Our studies contribute to understand the role of molecular chaperones and of PrP-related folding intermediates in "prion-like" conversion.
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http://dx.doi.org/10.1038/s41598-017-03969-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473912PMC
June 2017

Host defense peptide-derived privileged scaffolds for anti-infective drug discovery.

J Pept Sci 2017 Apr 12;23(4):303-310. Epub 2017 Jan 12.

CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145, Naples, Italy.

'Privileged scaffolds' are molecular frameworks which have been successfully exploited for small molecule drug discovery. Peptide privileged scaffolds, featuring a strictly conserved multiple-disulfide framework and high variability in the rest of the sequence, display a broad range of biological effects, including antimicrobial and antiviral activity. Unlike small molecules, however, the cost of manufacturing these peptides is high, and their synthesis challenging. We previously described a simplified privileged scaffold corresponding to the γ-core of human β-defensin-3 (HBD3). The γ-core is a common structural signature found in virtually all host defense peptides (HDPs) stabilized by multiple disulfides, and we showed that for HBD3, it represents the evolutionary starting point of the full-length molecule and, thus, is itself a primordial HDP. Accordingly, we showed that the peptide folded rapidly and was stable in human serum, and displayed many of the biological activities of HBD3. We report here that in addition to the previously reported antibacterial activity on planktonic bacteria, the γ-core peptide is active against biofilm formation and maturation. We also show that it is readily cell penetrant, like HBD3, although with a different mechanism, which is independent from CD98. Overall, the potency of the single-disulfide, 23-amino acid γ-core is comparable with the full-length peptide across the whole spectrum of examined properties, and the peptide is not toxic to human cells. The HBD3 γ-core peptide may therefore represent the first example of an economically viable lead peptide derived from a HDP privileged scaffold. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/psc.2962DOI Listing
April 2017

HMGA1 silencing reduces stemness and temozolomide resistance in glioblastoma stem cells.

Expert Opin Ther Targets 2016 10 8;20(10):1169-79. Epub 2016 Sep 8.

a Istituto di Endocrinologia ed Oncologia Sperimentale - CNR c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche , Università degli Studi di Napoli 'Federico II,' Naples , Italy.

Objective: Glioblastoma multiforme (GBM) develops from a small subpopulation of stem-like cells, which are endowed with the ability to self-renew, proliferate and give rise to progeny of multiple neuroepithelial lineages. These cells are resistant to conventional chemo- and radiotherapy and are hence also responsible for tumor recurrence. HMGA1 overexpression has been shown to correlate with proliferation, invasion, and angiogenesis of GBMs and to affect self-renewal of cancer stem cells from colon cancer. The role of HMGA1 in GBM tumor stem cells is not completely understood.

Research Design And Methods: We have investigated the role of HMGA1 in brain tumor stem cell (BTSC) self-renewal, stemness and resistance to temozolomide by shRNA- mediated HMGA1 silencing.

Results: We first report that HMGA1 is overexpressed in a subset of BTSC lines from human GBMs. Then, we show that HMGA1 knockdown reduces self-renewal, sphere forming efficiency and stemness, and sensitizes BTSCs to temozolomide. Interestingly, HMGA1 silencing also leads to reduced tumor initiation ability in vivo.

Conclusions: These results demonstrate a pivotal role of HMGA1 in cancer stem cell gliomagenesis and endorse HMGA1 as a suitable target for CSC-specific GBM therapy.
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http://dx.doi.org/10.1080/14728222.2016.1220543DOI Listing
October 2016

Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells.

Arch Biochem Biophys 2016 08 10;603:10-9. Epub 2016 May 10.

Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy. Electronic address:

Current models of G protein-coupled receptors (GPCRs) signaling describe binding of external agonists to cell surface receptors which, in turn, trigger several biological responses. New paradigms indicate that GPCRs localize to and signal at the nucleus, thus regulating distinct signaling cascades. The formyl-peptide receptor FPR2 belongs to the GPCR super-family and is coupled to PTX-sensitive Gi proteins. We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2 is expressed at nuclear level in CaLu-6 and AGS cells. Nuclear FPR2 is a functional receptor, since it participates in intra-nuclear signaling, as assessed by decreased G protein-FPR2 association and enhanced ERK2, c-Jun and c-Myc phosphorylation upon stimulation of intact nuclei with the FPR2 agonist, WKYMVm. We analyzed FPR2 sequence for the search of a nuclear localization sequence (NLS) and we found a stretch of basic aminoacids (227-KIHKK-231) in the third cytoplasmic loop of the receptor. We performed single (K230A) and multiple (H229A/K230A/K231A) mutagenesis of NLS. The constructs were individually overexpressed in HEK293 cells and immunofluorescence and western blot analysis showed that nuclear localization or translocation of FPR2 depends on the integrity of the H(229) and K(231) residues within the NLS.
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http://dx.doi.org/10.1016/j.abb.2016.05.006DOI Listing
August 2016

The 37/67 kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67 kDa LR cell surface localization and interaction with the cellular prion protein.

Sci Rep 2016 Apr 13;6:24457. Epub 2016 Apr 13.

Institut Pasteur, Unité de Trafic Membranaire et Pathogénèse, 75724 Paris CEDEX 15, France.

The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrP(C)) and to be required for pathological PrP (PrP(Sc)) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrP(C) interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrP(C) in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrP(C). Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrP(C) interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrP(C) on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrP(C) and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases.
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http://dx.doi.org/10.1038/srep24457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829897PMC
April 2016

Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients.

Am J Gastroenterol 2016 Jun 5;111(6):879-90. Epub 2016 Apr 5.

CEINGE-Biotecnologie Avanzate, Naples, Italy.

Objectives: Celiac disease (CD)-associated duodenal dysbiosis has not yet been clearly defined, and the mechanisms by which CD-associated dysbiosis could concur to CD development or exacerbation are unknown. In this study, we analyzed the duodenal microbiome of CD patients.

Methods: The microbiome was evaluated in duodenal biopsy samples of 20 adult patients with active CD, 6 CD patients on a gluten-free diet, and 15 controls by DNA sequencing of 16S ribosomal RNA libraries. Bacterial species were cultured, isolated and identified by mass spectrometry. Isolated bacterial species were used to infect CaCo-2 cells, and to stimulate normal duodenal explants and cultured human and murine dendritic cells (DCs). Inflammatory markers and cytokines were evaluated by immunofluorescence and ELISA, respectively.

Results: Proteobacteria was the most abundant and Firmicutes and Actinobacteria the least abundant phyla in the microbiome profiles of active CD patients. Members of the Neisseria genus (Betaproteobacteria class) were significantly more abundant in active CD patients than in the other two groups (P=0.03). Neisseria flavescens (CD-Nf) was the most abundant Neisseria species in active CD duodenum. Whole-genome sequencing of CD-Nf and control-Nf showed genetic diversity of the iron acquisition systems and of some hemoglobin-related genes. CD-Nf was able to escape the lysosomal compartment in CaCo-2 cells and to induce an inflammatory response in DCs and in ex-vivo mucosal explants.

Conclusions: Marked dysbiosis and an abundance of a peculiar CD-Nf strain characterize the duodenal microbiome in active CD patients thus suggesting that the CD-associated microbiota could contribute to the many inflammatory signals in this disorder.
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http://dx.doi.org/10.1038/ajg.2016.95DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4897008PMC
June 2016

An ancestral host defence peptide within human β-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule.

Sci Rep 2015 Dec 21;5:18450. Epub 2015 Dec 21.

CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italy.

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated "γ-core motif". We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents.
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http://dx.doi.org/10.1038/srep18450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685272PMC
December 2015

Novel mitochondrial protein interactors of immunoglobulin light chains causing heart amyloidosis.

FASEB J 2015 Nov 28;29(11):4614-28. Epub 2015 Jul 28.

*Amyloidosis Research and Treatment Center, Department of Molecular Medicine, **Department of Internal Medicine, Department of Cardiothoracic and Vascular Sciences, Laboratory of Experimental Cardiology for Cell and Molecular Therapy, University of Pavia, and Clinical Chemistry Laboratory, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy; Centro di Ricerca di Ingegneria Genetica (CEINGE)-Biotecnologie Avanzate, Naples, Italy; Department of Movement Sciences, Parthenope University of Naples, Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy; Department of Biology, University of Padua, Padua, Italy; and Department of Medicine, University of Cape Town, Cape Town, South Africa

In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into life-threatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiple myeloma without amyloidosis (the nonamyloidogenic/noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy.
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http://dx.doi.org/10.1096/fj.15-272179DOI Listing
November 2015

Binding of carbonic anhydrase IX to 45S rDNA genes is prevented by exportin-1 in hypoxic cells.

Biomed Res Int 2015 22;2015:674920. Epub 2015 Feb 22.

Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy ; CEINGE Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italy ; Associazione Culturale DiSciMuS RFC, 80026 Casoria, Italy.

Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45 S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45 S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45 S rDNA gene promoters.
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http://dx.doi.org/10.1155/2015/674920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352447PMC
January 2016

Pharmacological folding chaperones act as allosteric ligands of Frizzled4.

Nat Chem Biol 2015 Apr 9;11(4):280-6. Epub 2015 Mar 9.

Department of Pharmacy, University of Naples Federico II, Naples, Italy.

Upon binding, ligands can chaperone their protein targets by preventing them from misfolding and aggregating. Thus, an organic molecule that works as folding chaperone for a protein might be its specific ligand, and, similarly, the chaperone potential could represent an alternative readout in a molecular screening campaign toward the identification of new hits. Here we show that small molecules selected for acting as pharmacological chaperones on a misfolded mutant of the Frizzled4 (Fz4) receptor bind and modulate wild-type Fz4, representing what are to our knowledge the first organic ligands of this until-now-undruggable GPCR. The novelty and the advantages of the screening platform, the allosteric binding site addressed by these new ligands and the mechanism they use to modulate Fz4 suggest new avenues for development of inhibitors of the Wnt-β-catenin pathway and for drug discovery.
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http://dx.doi.org/10.1038/nchembio.1770DOI Listing
April 2015

Membrane protein 4F2/CD98 is a cell surface receptor involved in the internalization and trafficking of human β-Defensin 3 in epithelial cells.

Chem Biol 2015 Feb 29;22(2):217-28. Epub 2015 Jan 29.

CEINGE-Biotecnologie Avanzate S.c.a r.l., Via Gaetano Salvatore 486, 80145 Naples, Italy; IRCCS-Fondazione SDN, Via Emanuele Gianturco 113, 80142 Naples, Italy. Electronic address:

Human β-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human β-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3.
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http://dx.doi.org/10.1016/j.chembiol.2014.11.020DOI Listing
February 2015

New therapeutic perspectives in CCDC6 deficient lung cancer cells.

Int J Cancer 2015 May 24;136(9):2146-57. Epub 2014 Oct 24.

Istituto per l'Endocrinologia e l'Oncologia Sperimentale "Gaetano Salvatore", CNR, Napoli, Italy; Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Napoli, Italy.

Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.
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http://dx.doi.org/10.1002/ijc.29263DOI Listing
May 2015
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