Tesi di Dottorato

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    Modelli sperimentali cellulari e molecolari per la valutazione della tossicità di xenobiotici
    (2013-12-02) Peta, Valentina; Sisc, Diego; Indiveri, Cesare
    The aim of my thesis was to find two different experimental models to study cellular and molecular xenobiotics toxicity. In the first part of the present work we studied the interaction between two different plasma membrane transporters (OCTN2 and OCTN1), mercury reagents and heavy metals. Mercury and heavy metals in general cause toxic effects in many tissues interacting with protein cysteine (Cys) thiols. Transport systems represent critical targets of mercurials. Indeed, the majority of transport systems of higher eukaryotes containseveral Cys residues. One of the most up to date method of studying transport is the reconstitution of transportersin proteoliposomes. This method has been used as a useful approach to test the effect of HgCl2, methylmercury(MeHg) and Cadmium on the carnitine (OCTN2) transporter, extracted from rat kidney brush border membranes and reconstituted in liposomes by removing the detergent withhydrophobic chromatography columns, and the human organic cation transporter (OCTN1) overexpressed in E. coli, purified by Ni-chelating chromatography and reconstituted in liposomes by detergent removal with a batch-wise procedure.Transport was measured as [3H]carnitine uptake into proteoliposomescontaining carnitine (antiport reaction) in the case of rat OCTN2 and as [14C]tetraethylammonium uptake in the case of human OCTN1. Mercurials and heavy metals strongly inhibited the transport. Inhibition was reversed by1,4-dithioerythritol (DTE), L-cysteine (Cys), and N-acetyl-Lcysteine (NAC) indicating that it was caused by covalent reactionof mercurials and heavy metals with Cys residue(s) of OCTN2 and OCTN1.The presence of substrate prevented the inhibition in rat OCTN2 transporter indicating that the mercurial binding residue (Cys) is in the substrate binding site. No substrate protection was found in the case of the human OCTN1, so probably mercurial and heavy metal binding residue is away from substrate binding site. For the human OCTN1 we also tested the effect of chemical reagents which are known to form mixeddisulphides with proteins SH residues, MTS reagents. MTSEA exerted inhibition of transport very similar to those observed for the heavy metals and as the toxic compounds. To ascertain the involvement of Cys residues in the interaction of the human OCTN1 with the xenobiotics and to identify the possible target of the reagents, 7 mutants were prepared in which the seven cysteines present in the transmembrane domains or in the extracellular loop were mutated to alanine. An additional mutant lacking two Cys residues has also been constructed (C50A/C136A). We studies the dose-response curves of the transporter for each inhibitor, the mercury reagents showed similar behavior, both HgCl2 and MeHg strongly inhibited the WT and the mutants C81A, C113A, C236A, C270A and C374A. While a clear shift of the curves towards higher concentrations of the compounds was observed in the case of mutants C50A and C136A indicating a decrease of affinity of these mutants for the mercury reagents. The experiment on the double mutant C50A/C136A showed a nearly complete lack of inhibition by the two reagents demonstrating the two Cys residues were indeed the target of the mercury compounds. The homology model of the human OCTN1 confirms the experimental data obtained in this work, in fact the model shows that the two cysteine residues (Cys-50 and Cys-136) are exposed to the extracellular site of the plasma membrane and are accessible to sulfhydryl groups reagents. In the present work we studied, also, biomarkers expression and nephrotoxic effect induced by drugs in human kidney primary cells model. Kidney is a primary target of drug-induced toxicity. Toxic effects on the kidney related to drugs are both common and expected, given the kidney's roles in plasma filtration and maintenance of metabolic homeostasis. As such, glomerular, tubular and renal interstitial cells frequently are exposed to concentrations of drugs, which can induce changes in kidney function and structure. We tested three different drugs: 2-Bromoethanamine an analgesic, cisplatin a chemotherapeutic agent and cyclosporine a immunosuppressive agent. We found that after the addition of these drugs on human primary kidney cells there is an increase in the expression of two different biomarkers: Osteopontin (OPN) a distal tubule biomarker and KIM-1 a membrane glycoprotein expressed by proximal tubule cells after kidney injury. We also found the toxic concentrations after 24 hours of exposition to the three different drugs, we quantified the percentual of necrotic and apoptotic cells and we studied the toxicological effect of these drugs on cellular organelles like mitochondria. In conclusion proteoliposomes represent a suitable molecular model for studying the interaction of plasma membrane transport and toxic compounds, such as mercurials, and human primary cell culture is a valuable tool to study cell toxicity mechanisms of different drugs. Both the experimental models are a novel and potentially important tools in drug discovery and in the understanding of toxicity mechanism of xenobiotics compounds.
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    Heterologous over-expression of the human amino acid transportes LAT1-CD98 and ASCT2
    (2013-12-02) Pingitore, Piero; Pochinico, Lorena; Cerra, Maria Carmela
    Amino acid transport across the plasma membrane in mammalian cells is mediated by different transport systems such as the Na+-dependent systems A, ASC and N and the Na+- independent system L. Very interestingly some of these transporters such as ASCT2 and LAT1 are over-expressed in many tumors. Cancer cells, in fact, display enhanced need for amino acids and altered amino acid metabolism. Thus, structural and functional studies of these transporters are very important not only for characterization but also for applications in human therapy. Over-expression of the transport proteins is the starting point for obtaining purified transporters. Bacterial and/or yeast cell systems have been employed for this purpose, so far. LAT1 (SLC7A5) belongs to the system L which catalyze the transport of branched chain and aromatic amino acids. LAT1 is an heterodimer and its counterpart, CD98 (SLC3A2) protein, is probably involved in substrate recognition and membrane localization. Bacterial over-expression of the hLAT1 transporter has been performed using a screening strategy of E. coli strains transformed with several plasmid constructs. The best expression of the hLAT1 protein was achieved after cloning of the cDNA into pH6EX3 vector and transformation of Rosetta(DE3)pLysS cells. The hLAT1 protein was purified by Ni2+- chelating chromatography with a yield of about 3.5 mg/L. The cDNA coding for hCD98 was cloned in the pGEX-4T1 vector containing a N-terminal GST tag. Protein expression was obtained using the same bacterial strain above described. Differently from hLAT1, the GST-CD98 protein was soluble. hCD98 was obtained after thrombin treatment and separation by size exclusion chromatography, with a yield of 2 mg/L. ASCT2 (SLC1A5) belongs to the system ASC and has high affinity for Ala, Ser, Cys, Gln and Asn. E. coli revealed not suitable for expressing this protein. Thus, a different approach using P. pastoris was performed to produce the recombinant hASCT2 protein. After codon optimization for P. pastoris, the hASCT2 cDNA was cloned in the pPICZB expression vector carrying a C-terminal 6His-tag. For large protein production, the recombinant P. pastoris strain was grown in fermentors. The recombinant proteins was mainly localized to the membrane. After purification using Ni2+-NTA resin a yield of at least 10 mg/L was obtained. The procedure described can be now used for producing the three proteins in appropriate amounts for crystallization trials and functional studies.
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    Molecular mechanisms involved in the biological responses to estrogens and antiestrogens in cancer cell
    (2013-11-04) Madeo, Antonio; Passarino, Giuseppe; Maggiolini, Marcello
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    The G protein-coupled receptor GPR30 mediates estrogen signaling in cancer cells
    (2013-10-31) Albanito,Lidia; Maggiolini,Marcello; De Benedictis,Giovanna
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    Molecular effect on pre-mRNA tau alternative splicing of two novel intronic MAPT gene mutations associated to a sporadic case of frontotemporal dementia
    (2013-10-29) Vuono,Romina; Losso,Maria Adele; Bruni,Amalia Cecilia; De Benedictis, Giovanna
    Frontotemporal lobar degeneration (FTLD) is a heterogeneous syndrome encompassing different nosological entities characterized by behavioural and personality change, accompanied by deterioration of executive function, language and movement. Clinically FTLD results in at least three distinct syndromes: Frontotemporal dementia (FTD), Semantic dementia (SD) and Primary progressive aphasia (PPA), while the pathological classification is based on histopathological presence or absence of neuronal inclusions of tau and/or ubiquitin proteins accumulating in the neuronal/glial inclusions, being forms of FTLD differentiated in tau-positive, ubiquitin-positive and tau-negative. The most common clinical manifestation of FTLD is FTD, characterized by atrophy of the frontal and temporal lobes, with neuronal loss, gliosis and spongiosis of the superficial layers. FTD is mostly a presenile disorder showing changes in personality, impaired social conduct, emotional blunting, loss of insight, disinhibition, perseverative behaviour and hyperorality; cognitive deterioration, especially in language and in executive functions, appear later. Despite most cases of FTD are sporadic, approximately 10%-50% of FTD patients have a positive family history for dementia. Familial FTDL was associated to mutations in four genes: Microtubules associated protein tau (MAPT) and Progranulin (PGRN) genes that are responsible for the most genetic forms of FTD; instead, Valosin containing protein (VCP) gene is involved in rare forms of FTD with inclusion body myopathy and Paget’s disease of the bone and Charged multivescicolar body protein 2B (CHMP2B) is mutated in some families with a combination of FTD and Amyotrophic lateral sclerosis (ALS). Mutations in MAPT gene are responsible for 10%-20% of familial FTD. Alternative splicing of exons 2, 3 and 10 in MAPT pre m-RNA results in the expression of six isoforms. Exclusion or inclusion of Exon 10 gives rise to tau isoforms with three (tau3R, E10-) o four (tau4R, E10+) microtubule-binding repeats. In normal adult human brain the overall ratio of 3R to 4R tau is generally 1, whereas in fetal brain only the shortest tau isoform with 3R is expressed, indicating that tau expression is developmentally regulated.To date, 44 different potential pathogenic MAPT mutations have been reported, divided into two groups depending on the primary molecular mechanism involved: missense or deletion mutations that commonly modify tau interaction with microtubules and splicing mutations that affect the alternative splicing of exon 10, leading to changes of the ratio of 3R-tau/4R-tau. However, a third group of mutations exists that might have effects at protein and RNA levels. In the present study we report the molecular effect of two novel heterozygous MAPT gene mutations, a T to C transition at position -15 of intron 9 [T(-15)C] and an A to C transversion at position +4 of intron 10 (E10+4), identified in a patient with sporadic FTD, clinically and neuropathologically ascertained. Considering that both mutations are located in the splicing regulatory regions surrounding Exon 10, we analyzed their molecular effect on the alternative splicing of MAPT pre-mRNA in a minigene model system and in brain tissue. Semi-quantitative RT-PCR analyses, in minigene costructs and in brain tissue, have shown that the two novel mutations cause a novel Exon 10 splicing effect giving rise to a higher increase of mRNAs transcripts lacking Exon 10 (E10- or Tau3R) when compared with FTD-Ub+ control. Immunohistochemical and biochemical analyses on brain tissue evidenced neuronal and oligodendroglial tau deposits mostly made of Tau3R isoforms and an increased increased availability of shorter Tau3R isoform respectively. Data obtained with minigenes derived by the phenotipically healthy patient’s parents demonstrate that when the mutations are inherited in a non compound heterozygous condition the ratio of E10 including/E10 excluding transcripts is quite normal. Although the molecular mechanism underlying exon 10 splicing regulation remain to be completely elucidated, the exon 10 splice donor site is predicted to give rise to a RNA stem loop structure considered crucial for the quantitative regulation of exon 10 alternative splicing. Most of previously characterized mutations identified in the upper part of the stem loop strongly alter mRNA splicing by destabilizing the secondary structure, with a corresponding increase of E10 inclusion and 4RTau expression. Considering that the E10+4 mutation is located into the exon 10 splice donor site, we also investigated the effect of the E10+4 mutation on the thermodynamic stability of the RNA stem loop structure. Our data, based on bioinformatic prediction of the stem loop sequence thermostability and Ultraviolet Melting experiments demonstrated a strong increasing of stability in the stem-loop structure carrying the E10+4 mutation. This higher stability could be important for the skipping of exon 10, even though the E10+4 mutation alone is not able to give rise to a pathologic phenotype. We cannot exclude that the T to C transition, localized in a regulatory region upstream of exon 10, could also alter the binding of specific trans-splicing factors increasing the effect of the E10+4 mutation, giving rise when both mutation are present in the compound heterozygous condition (namely the FTD patient) to the E10 exclusion and the altered 4R/3R tau ratio observed. Thus, we can hypothesize a trans-acting regulatory effect of both mutations with known, or unknown splicing factors, which might have contributed to the very atypical clinical and pathological FTD phenotype of the patient.
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    Expression profiles of stress-responder nuclear genes in relationship to common mitochondrial DNA variability
    (2013-10-28) Taverna,Daniela; Bellizzi,Dina; De Benedictis,Giovanna