Publications by authors named "Marialuisa Casella"

12 Publications

  • Page 1 of 1

Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation.

Life Sci Alliance 2020 12 26;3(12). Epub 2020 Oct 26.

Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR), c/o Sapienza University, Rome, Italy

Hereditary Spastic Paraplegia (HSP) is a neurodegenerative disease most commonly caused by autosomal dominant mutations in the gene encoding the microtubule-severing protein spastin. We hypothesise that -HSP is attributable to reduced spastin function because of haploinsufficiency; thus, therapeutic approaches which elevate levels of the wild-type spastin allele may be an effective therapy. However, until now, how spastin levels are regulated is largely unknown. Here, we show that the kinase HIPK2 regulates spastin protein levels in proliferating cells, in differentiated neurons and in vivo. Our work reveals that HIPK2-mediated phosphorylation of spastin at S268 inhibits spastin K48-poly-ubiquitination at K554 and prevents its neddylation-dependent proteasomal degradation. In a spastin RNAi neuronal cell model, overexpression of HIPK2, or inhibition of neddylation, restores spastin levels and rescues neurite defects. Notably, we demonstrate that spastin levels can be restored pharmacologically by inhibiting its neddylation-mediated degradation in neurons derived from a spastin mouse model of HSP and in patient-derived cells, thus revealing novel therapeutic targets for the treatment of -HSP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.26508/lsa.202000799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652396PMC
December 2020

Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors.

Front Immunol 2020 13;11:262. Epub 2020 Mar 13.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity and . Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101CD56 nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFβ), respectively. Furthermore, NKEVs increased the CD56 NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101CD56 exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.00262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082405PMC
March 2020

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site.

PLoS One 2018 15;13(2):e0192651. Epub 2018 Feb 15.

CNR Institute for Molecular Recognition, Roma Italy.

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813964PMC
April 2018

Proteomic and functional analyses reveal pleiotropic action of the anti-tumoral compound NBDHEX in Giardia duodenalis.

Int J Parasitol Drugs Drug Resist 2017 08 29;7(2):147-158. Epub 2017 Mar 29.

Department of Infectious Diseases, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy. Electronic address:

Giardiasis, a parasitic diarrheal disease caused by Giardia duodenalis, affects one billion people worldwide. Treatment relies only on a restricted armamentarium of drugs. The disease burden and the increase in treatment failure highlight the need for novel, safe and well characterized drug options. The antitumoral compound NBDHEX is effective in vitro against Giardia trophozoites and inhibits glycerol-3-phosphate dehydrogenase. Aim of this work was to search for additional NBDHEX protein targets. The intrinsic NBDHEX fluorescence was exploited in a proteomic analysis to select and detect modified proteins in drug treated Giardia. In silico structural analysis, intracellular localization and functional assays were further performed to evaluate drug effects on the identified targets. A small subset of Giardia proteins was covalently bound to the drug at specific cysteine residues. These proteins include metabolic enzymes, e.g. thioredoxin reductase (gTrxR), as well as elongation factor 1B-γ (gEF1Bγ), and structural proteins, e.g. α-tubulin. We showed that NBDHEX in vitro binds to recombinant gEF1Bγ and gTrxR, but only the last one could nitroreduce NBDHEX leading to drug modification of gTrxR catalytic cysteines, with concomitant disulphide reductase activity inhibition and NADPH oxidase activity upsurge. Our results indicate that NBDHEX reacts with multiple targets whose roles and/or functions are specifically hampered. In addition, NBDHEX is in turn converted to reactive intermediates extending its toxicity. The described NBDHEX pleiotropic action accounts for its antigiardial activity and encourages the use of this drug as a promising alternative for the future treatment of giardiasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpddr.2017.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377010PMC
August 2017

Mass spectrometry detection of fraudulent use of cow whey in water buffalo, sheep, or goat Italian ricotta cheese.

Food Chem 2016 Apr 14;197 Pt B:1240-8. Epub 2015 Nov 14.

Ministry of Agriculture Food and Forest Policies, Department of Central Inspectorate for Food and Feed Quality Protection and Fraud Repression, Central Laboratory of Rome, Via del Fornetto, 85, 00149 Rome, Italy. Electronic address:

Ricotta cheese is a typical Italian product, made with whey from various species, including cow, buffalo, sheep, and goat. Ricotta cheese nominally manufactured from the last three species may be fraudulently produced using the comparatively cheaper cow whey. Exposing such food frauds requires a reliable analytical method. Despite the extensive similarities shared by whey proteins of the four species, a mass spectrometry-based analytical method was developed that exploits three species-specific peptides derived from β-lactoglobulin and α-lactalbumin. This method can detect as little as 0.5% bovine whey in ricotta cheese from the other three species. Furthermore, a tight correlation was found (R(2)>0.99) between cow whey percentages and mass spectrometry measurements throughout the 1-50% range. Thus, this method can be used for forensic detection of ricotta cheese adulteration and, if properly validated, to provide quantitative evaluations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.foodchem.2015.11.073DOI Listing
April 2016

Gaucher disease due to saposin C deficiency is an inherited lysosomal disease caused by rapidly degraded mutant proteins.

Hum Mol Genet 2014 Nov 12;23(21):5814-26. Epub 2014 Jun 12.

Department of Haematology, Oncology and Molecular Medicine,

Saposin (Sap) C is an essential cofactor for the lysosomal degradation of glucosylceramide (GC) by glucosylceramidase (GCase) and its functional impairment underlies a rare variant form of Gaucher disease (GD). Sap C promotes rearrangement of lipid organization in lysosomal membranes favoring substrate accessibility to GCase. It is characterized by six invariantly conserved cysteine residues involved in three intramolecular disulfide bonds, which make the protein remarkably stable to acid environment and degradation. Five different mutations (i.e. p.C315S, p.342_348FDKMCSKdel, p.L349P, p.C382G and p.C382F) have been identified to underlie Sap C deficiency. The molecular mechanism by which these mutations affect Sap C function, however, has not been delineated in detail. Here, we characterized biochemically and functionally four of these gene lesions. We show that all Sap C mutants are efficiently produced, and exhibit lipid-binding properties, modulatory behavior on GCase activity and subcellular localization comparable with those of the wild-type protein. We then delineated the structural rearrangement of these mutants, documenting that most proteins assume diverse aberrant disulfide bridge arrangements, which result in a substantial diminished half-life, and rapid degradation via autophagy. These findings further document the paramount importance of disulfide bridges in the stability of Sap C and provide evidence that accelerated degradation of the Sap C mutants is the underlying pathogenetic mechanism of Sap C deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddu299DOI Listing
November 2014

Carbon monoxide signaling in human red blood cells: evidence for pentose phosphate pathway activation and protein deglutathionylation.

Antioxid Redox Signal 2014 Jan 2;20(3):403-16. Epub 2013 Aug 2.

1 Department of Cell Biology and Neurosciences, Sections of Biomarkers in Degenerative Diseases, Istituto Superiore di Sanità , Rome, Italy .

Aims: The biochemistry underlying the physiological, adaptive, and toxic effects of carbon monoxide (CO) is linked to its affinity for reduced transition metals. We investigated CO signaling in the vasculature, where hemoglobin (Hb), the CO most important metal-containing carrier is highly concentrated inside red blood cells (RBCs).

Results: By combining NMR, MS, and spectrophotometric techniques, we found that CO treatment of whole blood increases the concentration of reduced glutathione (GSH) in RBC cytosol, which is linked to a significant Hb deglutathionylation. In addition, this process (i) does not activate glycolytic metabolism, (ii) boosts the pentose phosphate pathway (PPP), (iii) increases glutathione reductase activity, and (iv) decreases oxidized glutathione concentration. Moreover, GSH concentration was partially decreased in the presence of 2-deoxyglucose and the PPP antagonist dehydroepiandrosterone. Our MS results show for the first time that, besides Cys93, Hb glutathionylation occurs also at Cys112 of the β-chain, providing a new potential GSH source hitherto unknown.

Innovation: This work provides new insights on the signaling and antioxidant-boosting properties of CO in human blood, identifying Hb as a major source of GSH release and the PPP as a metabolic mechanism supporting Hb deglutathionylation.

Conclusions: CO-dependent GSH increase is a new RBC process linking a redox-inactive molecule, CO, to GSH redox signaling. This mechanism may be involved in the adaptive responses aimed to counteract stress conditions in mammalian tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ars.2012.5102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894680PMC
January 2014

Novel pharmacokinetic behavior of intravenous busulfan in children with thalassemia undergoing hematopoietic stem cell transplantation: a prospective evaluation of pharmacokinetic and pharmacodynamic profile with therapeutic drug monitoring.

Blood 2010 Jun 17;115(22):4597-604. Epub 2010 Mar 17.

International Center for Transplantation in Thalassemia and Sickle Cell Anemia, Mediterranean Institute of Hematology, Policlinico Tor Vergata, Rome, Italy.

We prospectively studied the pharmacokinetics (PK) and clinical outcomes of intravenous busulfan (Bu) in 71 children with preexisting liver damage who underwent hematopoietic stem cell transplantation for thalassemia. Intravenous Bu was administered every 6 hours as part of a conditioning regimen with PK-based dose adjustment to target a conservative area under the concentration-versus-time curve (AUC) range (900-1350 microMol*min). The first-dose Bu clearance (CL) was significantly higher than the subsequent daily CL that remained unchanged in the ensuing days. One-third of patients required dose escalation based on dose 1 AUC, whereas dose reduction was needed in the subsequent days. At doses 5, 9, and 13, 78%, 81%, and 87% of patients, respectively, achieved the target range of AUC. A population PK analysis confirmed that the first-dose CL was 20% higher and that body weight was the most important covariate to explain PK variability. Patients with variant GSTA1*B had a 10% lower Bu CL than wild-type. These results suggest that the disease-specific behavior of intravenous Bu PK should be considered for PK-guided dose adjustment in patients with thalassemia, and the use of a conservative AUC range resulted in low toxicity, good engraftment, and good survival rate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2010-01-265405DOI Listing
June 2010

PCB, PCDD and PCDF contamination of food of animal origin as the effect of soil pollution and the cause of human exposure in Brescia.

Chemosphere 2009 Jun 5;76(2):278-85. Epub 2009 Apr 5.

Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

In Brescia a PCB production plant polluted soil and forage of the surrounding fields and caused a significant contamination of meat and milk of the cattle fed with local forage. This in turn induced elevated blood levels of PCDDs, PCDFs and PCBs in the consumers. The contamination levels and profiles measured in the perirenal fat, in the liver and in the milk of the overall 28 contaminated bovines are reported. TEQ levels varied from 30 to 81 pg WHO(2005)-TEQ g(-1) (38-103 pg WHO(1997)-TEQ) for perirenal fat, from 107 to 138 pg WHO(2005)-TEQ g(-1) fat (128-168 pg WHO(1997)-TEQ) for liver and from 45 to 50 pg WHO(2005)-TEQg(-1) fat (56-65pg WHO(1997)-TEQ) for milk; all these values are roughly tenfold higher than the European limits. Non-ortho dioxin-like (dl)PCBs are by far the largest contributors to TEQ and PCDF contribution also largely prevail over PCDD's; both these features are also present in both the contaminated forages and in the serum of consumers of contaminated food. The indicator PCB levels are in the following ranges: 226-664 ng g(-1) for perirenal fat; 929-1822 ng g(-1) fat for liver; 183-477 ng g(-1) fat for milk; their level is about 100 times higher than the regional background. The liver samples displayed an overall TEQ several times higher than the perirenal fat from either the same animal or the same pool of animals; the increase in liver concentration was significantly higher for PCDD and PCDF congeners than for dlPCBs, and it was maximum for OCCD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chemosphere.2009.03.002DOI Listing
June 2009

PCDD/F and PCB in human serum of differently exposed population groups of an Italian city.

Chemosphere 2008 Aug 2;73(1 Suppl):S228-34. Epub 2008 Jun 2.

Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

A chemical plant located in Brescia, an industrial city in North-Western Italy, produced polychlorobiphenyls (PCBs) during a 30-50 year period, causing widespread pollution of the surrounding agricultural area. This area contains several small farms, which principally produce veal meat for private consumption of the farmers' families. The pollution went undiscovered for many years, during which period contaminated food was regularly consumed. This paper reports the polychlorodibenzodioxin (PCDD), polychlorodibenzofuran (PCDF) and PCB levels of a serum sample pooled from the consumers of contaminated food, compared to six population groups of the city of Brescia. Four of these groups were selected in order to represent, respectively, the local general population and the residents of three zones of the polluted area, while the last two groups represented, respectively, the present and the former workers of the plant. One human milk sample from one of the consumers of contaminated food was also analyzed. Results show that the consumers of the contaminated food and the former workers of the plant display considerably higher levels than all other groups. The levels of general population and of all other groups were generally similar both to each other and to the range of literature values for unexposed populations. The respective contribution of PCDDs, PCDFs, mono-ortho and non-ortho PCBs (dioxin-like PCBs) to (Toxicity Equivalents) TEQ of the population groups of this study were also compared to literature data: the two groups with a high contamination level, together with the human milk sample, displayed a higher incidence of mono-ortho PCBs and a lower contribution of PCDD, possibly correlated with the source of contamination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chemosphere.2008.01.081DOI Listing
August 2008

A study on PCB, PCDD/PCDF industrial contamination in a mixed urban-agricultural area significantly affecting the food chain and the human exposure. Part I: soil and feed.

Chemosphere 2007 Apr 17;67(9):1822-30. Epub 2007 Jan 17.

Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy.

This study deals with a PCB, PCDD and PCDF contamination in Brescia, a city in the North-West of Italy, affecting an area with about 11000 inhabitants. The area is close to an industrial plant that produced, in total, some 31,000 ton of PCB. A relevant part of the polluted area is agricultural soil, where cattle were fed with polluted forage and farmers were consuming their own products, so that contamination led eventually to human exposure. Total levels of PCDD/Fs varied from 8 to 592 pgTE(WHO)/g for soil samples and when the dioxin-like PCBs (dl-PCBs) are included, the levels varied from 14.6 to 1033.7 pgTE(WHO)/g. In several cases, the legal limit was exceeded by more than one order of magnitude, with the highest contamination in some agricultural areas and in the surrounding zones. For the forage samples, total levels of PCDD/Fs varied from 0.29 to 2.04 pgTE(WHO)/g and, when dl-PCBs are included, this range increased from 2.04 to 4.75 pgTE(WHO)/g. PCB contamination of the forage through vapor condensation seemed to be relevant. The toxic contribution of dl-PCBs is always relevant and must be considered for risk management. The main component of the contamination source is probably a heavy PCB mixture, such as Aroclor 1262. The study dealt generally with the contamination transfer of PCBs, PCDDs and PCDFs from soil up to humans across the food chain. Results on soils and forages are shown, while measurements concerning the contamination of the animals fed with contaminated forage, and the exposure of the farmers (through human serum analyses), as compared to general population, will be reported in a dedicated paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chemosphere.2006.05.124DOI Listing
April 2007

Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules.

Mol Biol Cell 2006 Mar 11;17(3):1388-98. Epub 2006 Jan 11.

Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Germany.

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.e05-08-0708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1382326PMC
March 2006