Publications by authors named "Marco Gaspari"

78 Publications

Mass Spectrometry-Based Glycoproteomics and Prostate Cancer.

Int J Mol Sci 2021 May 14;22(10). Epub 2021 May 14.

Research Centre for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, 88100 Catanzaro, Italy.

Aberrant glycosylation has long been known to be associated with cancer, since it is involved in key mechanisms such as tumour onset, development and progression. This review will focus on protein glycosylation studies in cells, tissue, urine and serum in the context of prostate cancer. A dedicated section will cover the glycoforms of prostate specific antigen, the molecule that, despite some important limitations, is routinely tested for helping prostate cancer diagnosis. Our aim is to provide readers with an overview of mass spectrometry-based glycoproteomics of prostate cancer. From this perspective, the first part of this review will illustrate the main strategies for glycopeptide enrichment and mass spectrometric analysis. The molecular information obtained by glycoproteomic analysis performed by mass spectrometry has led to new insights into the mechanism linking aberrant glycosylation to cancer cell proliferation, migration and immunoescape.
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http://dx.doi.org/10.3390/ijms22105222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156230PMC
May 2021

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis.

J Vis Exp 2021 05 8(171). Epub 2021 May 8.

Research Centre for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro.

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.
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http://dx.doi.org/10.3791/62512DOI Listing
May 2021

LC-MALDI-TOF ISD MS analysis is an effective, simple and rapid method of investigation for histones characterization: Application to EBV lymphoblastoid cell lines.

J Mass Spectrom 2021 Feb 20;56(5):e4712. Epub 2021 Feb 20.

National Research Council, Institute for Biomedical Research and Innovation (IRIB), Cosenza, Italy.

This contribution is the result of our progressive engagement to develop and to apply a top-down liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (LC-MALDI-TOF) analysis for the histone post-translational modifications (PTMs) and variants characterization, mainly in order to provide comprehensive and fast results. The histone post-translational modifications and the differential expression of the histone variants play an essential role both in the DNA packaging mechanism in chromosomes and in the regulation of gene expression in different cellular processes, also in response to molecular agents of environmental origin. This epigenetic mechanism is widely studied in different field such as cellular differentiation, development and in the understanding of mechanisms underlying diseases. The characterization of histone PTMs has traditionally performed by antibodies-based assay, but immunological methods have significant limits, and today systems that use mass spectrometry are increasingly employed. We evaluated an in-source decay (ISD) analysis for the histone investigation on human lymphoblastoid cells, and by this approach, we were able to identify and quantify several PTMs such as the di-methylation in the lysine 20 and the acetylation in the lysine 16 in H4 and the mono-methylation, di-methylation and trimethylations at K9 of the histone H3.1. Moreover, we detected and quantified in the same H2B spectrum the prevalent H2B 1C/2E type but also the minor H2B 1D, 1M and 1B/1L/1N, 1O/2F, 1J/1K variants. In this work, we show that MALDI-ISD represents an excellent methodology to obtain global information on histone PTMs and variants from cells in culture, with rapidity and simplicity of execution. Finally, this is a useful approach to get label-free relative quantitative data of histone variants and PTMs.
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http://dx.doi.org/10.1002/jms.4712DOI Listing
February 2021

Cytoplasmic cleavage of IMPA1 3' UTR is necessary for maintaining axon integrity.

Cell Rep 2021 02;34(8):108778

MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK. Electronic address:

The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.
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http://dx.doi.org/10.1016/j.celrep.2021.108778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918530PMC
February 2021

Proteomic analysis from skin swabs reveals a new set of proteins identifying skin impairment in atopic dermatitis.

Exp Dermatol 2021 Jun 23;30(6):811-819. Epub 2021 Jan 23.

Department of Health Science, 'Magna Graecia' University of Catanzaro, Catanzaro, Italy.

Atopic Dermatitis (AD) is a common inflammatory skin disease characterized by skin and systemic inflammation, and barrier dysfunction. Herein, we investigate the proteomic profile of AD skin barrier to identify a unique signature with an easy-performed sampling approach. We enrolled 8 moderate-to-severe AD patients and 8 age- and gender-matched healthy controls. Swabs were obtained from non-lesional skin of retroauricular area and antecubital fold. Peptide mixtures obtained through protein precipitation and in-solution digestion were analysed using NanoLC-MS/MS. Label-free quantification and statistical analysis were conducted in MaxQuant and Perseus. Bioinformatics analysis was performed using Gene Ontology and STRING. We identified 908 proteins and 35 differentially expressed proteins were selected (fold change 2, FDR < 0.05). Particularly, AD skin showed downregulation of skin hydration factors, structural and epidermal proteins, abnormalities in protease-proteasome complex and lipid metabolism profile. Imbalance of antioxidant and inflammatory processes, along with TDRD15 upregulation was also observed. Our result showed partial overlap with skin biopsy/tape-strips studies, showing the reliability of our sampling approach which could be an easier method of detection of hallmark barrier proteins in AD. Furthermore, we displayed a new differentially expressed set of proteins, not yet explored in AD which can have a potential role in AD pathomechanisms.
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http://dx.doi.org/10.1111/exd.14276DOI Listing
June 2021

A critical comparison of three MS-based approaches for quantitative proteomics analysis.

J Mass Spectrom 2021 Jan 31;56(1):e4669. Epub 2020 Oct 31.

Research Centre for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Catanzaro, Italy.

MS-based proteomics is expanding its role as a routine tool for biological discovery. Nevertheless, the task of accurately and precisely quantifying thousands of analytes in a single experiment remains challenging. In this study, the diagnostic accuracy of three popular data-dependent methods for protein relative quantification (label-free [LF], dimethyl labelling [DML] and tandem mass tags [TMT]) has been assessed using a mixed species proteome (three species) and five experimental replicates per condition. Data were produced using a quadrupole-Orbitrap mass spectrometer and analysed using a single platform (the MaxQuant/Perseus software suite). The whole comparative analysis was repeated three times over a period of 6 months, in order to assess the consistency of the reported findings. As expected, label-based methods reproducibly provided a lower false positives rate, whereas TMT and LF performed similarly, and significantly better than DML, in terms of proteome coverage using the same instrument time. Although parameters like proteome coverage and precision were consistent in between replicates, other parameters like sensitivity, intended as the capacity of correctly classifying true positives (regulated proteins), were found to be less reproducible, especially at challenging fold-changes (1.5). Collectively, data suggest that an increased interest in data reproducibility would be desirable in the quantitative proteomics field.
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http://dx.doi.org/10.1002/jms.4669DOI Listing
January 2021

miR-21 antagonism abrogates Th17 tumor promoting functions in multiple myeloma.

Leukemia 2021 03 6;35(3):823-834. Epub 2020 Jul 6.

Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy.

Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.
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http://dx.doi.org/10.1038/s41375-020-0947-1DOI Listing
March 2021

Human adipose-derived mesenchymal stem cell-conditioned medium ameliorates polyneuropathy and foot ulceration in diabetic BKS db/db mice.

Stem Cell Res Ther 2020 05 1;11(1):168. Epub 2020 May 1.

Center for Regenerative Medicine, School of Medicine Clínica Alemana-Universidad del Desarrollo, Av. Las Condes 12438, Lo Barnechea, Santiago, Chile.

Background: Diabetic polyneuropathy (DPN) is the most common and early developing complication of diabetes mellitus, and the key contributor for foot ulcers development, with no specific therapies available. Different studies have shown that mesenchymal stem cell (MSC) administration is able to ameliorate DPN; however, limited cell survival and safety reasons hinder its transfer from bench to bedside. MSCs secrete a broad range of antioxidant, neuroprotective, angiogenic, and immunomodulatory factors (known as conditioned medium), which are all decreased in the peripheral nerves of diabetic patients. Furthermore, the abundance of these factors can be boosted in vitro by incubating MSCs with a preconditioning stimulus, enhancing their therapeutic efficacy. We hypothesize that systemic administration of conditioned medium derived from preconditioned MSCs could reverse DPN and prevent foot ulcer formation in a mouse model of type II diabetes mellitus.

Methods: Diabetic BKS db/db mice were treated with systemic administration of conditioned medium derived from preconditioned human MSCs; conditioned medium derived from non-preconditioned MSCs or vehicle after behavioral signs of DPN was already present. Conditioned medium or vehicle administration was repeated every 2 weeks for a total of four administrations, and several functional and structural parameters characteristic of DPN were evaluated. Finally, a wound was made in the dorsal surface of both feet, and the kinetics of wound closure, re-epithelialization, angiogenesis, and cell proliferation were evaluated.

Results: Our molecular, electrophysiological, and histological analysis demonstrated that the administration of conditioned medium derived from non-preconditioned MSCs or from preconditioned MSCs to diabetic BKS db/db mice strongly reverts the established DPN, improving thermal and mechanical sensitivity, restoring intraepidermal nerve fiber density, reducing neuron and Schwann cell apoptosis, improving angiogenesis, and reducing chronic inflammation of peripheral nerves. Furthermore, DPN reversion induced by conditioned medium administration enhances the wound healing process by accelerating wound closure, improving the re-epithelialization of the injured skin and increasing blood vessels in the wound bed in a skin injury model that mimics a foot ulcer.

Conclusions: Studies conducted indicate that MSC-conditioned medium administration could be a novel cell-free therapeutic approach to reverse the initial stages of DPN, avoiding the risk of lower limb amputation triggered by foot ulcer formation and accelerating the wound healing process in case it occurs.
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http://dx.doi.org/10.1186/s13287-020-01680-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195803PMC
May 2020

Comprehensive proteogenomic analysis of human embryonic and induced pluripotent stem cells.

J Cell Mol Med 2019 08 25;23(8):5440-5453. Epub 2019 Jun 25.

Research Center for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.

Although the concepts of somatic cell reprogramming and human-induced pluripotent stem cells (hiPSCs) generation have undergone several analyses to validate the usefulness of these cells in research and clinic, it remains still controversial whether the hiPSCs are equivalent to human embryonic stem cells (hESCs), pointing to the need of further characterization for a more comprehensive understanding of pluripotency. Most of the experimental evidence comes from the transcriptome analysis, while a little is available on protein data, and even less is known about the post-translational modifications. Here, we report a combined strategy of mass spectrometry and gene expression profiling for proteogenomic analysis of reprogrammed and embryonic stem cells. The data obtained through this integrated, multi-"omics" approach indicate that a small, but still significant, number of distinct pathways is enriched in reprogrammed versus embryonic stem cells, supporting the view that pluripotency is an extremely complex, multifaceted phenomenon, with peculiarities that are characteristic of each cell type.
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http://dx.doi.org/10.1111/jcmm.14426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6653499PMC
August 2019

Investigating the Anticancer Activity of Isatin/Dihydropyrazole Hybrids.

ACS Med Chem Lett 2019 Apr 18;10(4):571-576. Epub 2018 Dec 18.

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy.

A series of isatin-dihydropyrazole hybrids have been synthesized in order to assess their potential as anticancer agents. In particular, 12 compounds were evaluated for their antiproliferative activity toward A549, IGR39, U87, MDA-MB-231, MCF-7, BT474, BxPC-3, SKOV-3, and H1299 cell lines, and human foreskin fibroblasts. Four compounds exhibited interesting antiproliferative activity and were further examined to determine their EC values toward a panel of selected tumor cell lines. The best compounds were then investigated for their induced mechanism of cell death. Preliminary structure-activity relationship indicates that the presence of a substituent such as a chlorine atom or a methyl moiety in position 5 of the isatin nucleus is beneficial for the antitumor activity. proved the most promising compound within the studied series with EC values ranging from 0.01 to 0.38 μM.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466827PMC
April 2019

Regulation of NGF Signaling by an Axonal Untranslated mRNA.

Neuron 2019 05 7;102(3):553-563.e8. Epub 2019 Mar 7.

MRC Laboratory for Molecular Cell Biology, University College London, London, UK. Electronic address:

Neurons are extraordinarily large and highly polarized cells that require rapid and efficient communication between cell bodies and axons over long distances. In peripheral neurons, transcripts are transported along axons to growth cones, where they are rapidly translated in response to extrinsic signals. While studying Tp53inp2, a transcript highly expressed and enriched in sympathetic neuron axons, we unexpectedly discovered that Tp53inp2 is not translated. Instead, the transcript supports axon growth in a coding-independent manner. Increasing evidence indicates that mRNAs may function independently of their coding capacity; for example, acting as a scaffold for functionally related proteins. The Tp53inp2 transcript interacts with the nerve growth factor (NGF) receptor TrkA, regulating TrkA endocytosis and signaling. Deletion of Tp53inp2 inhibits axon growth in vivo, and the defects are rescued by a non-translatable form of the transcript. Tp53inp2 is an atypical mRNA that regulates axon growth by enhancing NGF-TrkA signaling in a translation-independent manner.
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http://dx.doi.org/10.1016/j.neuron.2019.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509357PMC
May 2019

Fhit-Fdxr interaction in the mitochondria: modulation of reactive oxygen species generation and apoptosis in cancer cells.

Cell Death Dis 2019 02 15;10(3):147. Epub 2019 Feb 15.

Department of Cancer Biology and Genetics, The Ohio State University Comprehensive Cancer Center, Columbus, OH, 43210, USA.

Fhit protein is lost in cancers of most, perhaps all, cancer types; when restored, it can induce apoptosis and suppress tumorigenicity, as shown in vitro and in mouse tumor models in vivo. Following protein cross-linking and proteomics analyses, we characterized a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes the heat-shock chaperonin pair, HSP60/10, which is likely involved in importing Fhit into the mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin, in electron transport chain complex III. Overexpression of Fhit protein in Fhit-deficient cancer cells modulates the production of intracellular reactive oxygen species, causing increased ROS, following peroxide treatment, with subsequent increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape ROS overproduction and ROS-induced apoptosis, likely carrying oxidative damage. Thus, characterization of Fhit-interacting proteins has identified direct effectors of a Fhit-mediated apoptotic signal pathway that is lost in many cancers. This is of translational interest considering the very recent emphasis in a number of high-profile publications, concerning the role of oxidative phosphorylation in the treatment of human cancers, and especially cancer stem cells that rely upon oxidative phosphorylation for survival. Additionally, we have shown that cells from a Fhit-deficient lung cancer cell line, are sensitive to killing by exposure to atovaquone, thought to act as a selective oxidative phosphorylation inhibitor by targeting the CoQ10 dependence of the mitochondrial complex III, while the Fhit-expressing sister clone is resistant to this treatment.
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http://dx.doi.org/10.1038/s41419-019-1414-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377664PMC
February 2019

High-throughput detection of low abundance sialylated glycoproteins in human serum by TiO enrichment and targeted LC-MS/MS analysis: application to a prostate cancer sample set.

Anal Bioanal Chem 2019 Jan 28;411(3):755-763. Epub 2018 Nov 28.

Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Campus "S. Venuta", Viale Europa, Loc. Germaneto, 88100, Catanzaro, Italy.

Glycopeptide enrichment can be a strategy to allow the detection of peptides belonging to low abundance proteins in complex matrixes such as blood serum or plasma. Though several glycopeptide enrichment protocols have shown excellent sensitivities in this respect, few reports have demonstrated the applicability of these methods to relatively large sample cohorts. In this work, a fast protocol based on TiO enrichment and highly sensitive mass spectrometric analysis by Selected Reaction Monitoring (SRM) has been applied to a cohort of serum samples from prostate cancer and benign prostatic hyperplasia patients in order to detect low abundance proteins in a single LC-MS/MS analysis in nanoscale format, without immunodepletion or peptide fractionation. A peptide library of over 700 formerly N-glycosylated peptides was created by data dependent analysis. Then, 16 medium to low abundance proteins were selected for detection by single injection LC-MS/MS based on selected-reaction monitoring. Results demonstrated the consistent detection of the low-level proteins under investigation. Following label-free quantification, four proteins (Adipocyte plasma membrane-associated protein, Periostin, Cathepsin D and Lysosome-associated membrane glycoprotein 2) were found significantly increased in prostate cancer sera compared to the control group. Graphical abstract ᅟ.
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http://dx.doi.org/10.1007/s00216-018-1497-5DOI Listing
January 2019

On-Tissue Hydrogel-Mediated Nondestructive Proteomic Characterization: Application to fr/fr and FFPE Tissues and Insights for Quantitative Proteomics Using a Case of Cardiac Myxoma.

Proteomics Clin Appl 2019 01 12;13(1):e1700167. Epub 2018 Nov 12.

Research Center for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Campus "S. Venuta,", Viale Europa, Loc. Germaneto, 88100 Catanzaro, Italy.

Purpose: The application of a methodology for quantitative protein analysis from formalin-fixed and paraffin-embedded (FFPE) tissue by using hydrogels. Miniaturized polymeric gels are placed onto histologically defined tissue regions in order to perform localized digestion for bottom-up proteomics. Hydrogel-extracted peptides are then labeled with tandem mass tags (TMT) reagents for relative protein quantification. A cardiac myxoma biopsy is used.

Experimental Design: Multiple hydrogels, incorporating the proteolytic enzyme trypsin, are placed on serial tissue sections, and processed for digestion and TMT derivatization. SCX fractionation before LC-MS/MS analysis and bioinformatics analysis are carried out.

Results: Two histologically different areas on both FFPE and frozen sections of the same cardiac myxoma biopsy are compared. In total, 1949 (FFPE) and 2491 (frozen) proteins are identified, with a total overlap of 56%. The quantitative comparison highlighted 15 (FFPE) and 138 (frozen) differentially expressed proteins between myxoma regions.

Conclusion: The methodology successfully detects numerous protein signals from FFPE and frozen specimens and is able to differentiate between tissue regions. A fast and reliable tissue preparation for quantitative protein analysis by minimum sample manipulation is developed. This offers an option for on-tissue proteomics analysis while preserving the inherent spatial information on the tissue.
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http://dx.doi.org/10.1002/prca.201700167DOI Listing
January 2019

Tuning the Dual Inhibition of Carbonic Anhydrase and Cyclooxygenase by Dihydrothiazole Benzensulfonamides.

ACS Med Chem Lett 2018 Oct 17;9(10):1045-1050. Epub 2018 Sep 17.

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy.

A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides () was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOX-2 isozyme, indicating that 2,3,4-trisubstituted 2,3-dihydrothiazoles are a promising scaffold for the inhibition of hCA isozymes and of hCOX-2 enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enzyme families. resulted as the best performing compound toward both enzyme families and exhibited preferential activity toward hCA XII and hCOX-2 isozymes.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187410PMC
October 2018

Targeting Tumor Associated Carbonic Anhydrases IX and XII: Highly Isozyme Selective Coumarin and Psoralen Inhibitors.

ACS Med Chem Lett 2018 Jul 6;9(7):725-729. Epub 2018 Jun 6.

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy.

A small library of psoralen carboxylic acids and their corresponding benzenesulfonamide derivatives were designed and synthesized to evaluate their activity and selectivity toward tumor associated human carbonic anhydrase (hCA) isoforms IX and XII. Both psoralen acids and sulfonamides exhibited potent inhibition of IX and XII isozymes in the nanomolar concentration range. However, psoralen acids resulted as the most selective in comparison with the corresponding benzenesulfonamide derivatives. Our data indicate that the psoralen scaffold is a promising starting point for the design of highly selective tumor associated hCA inhibitors.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047168PMC
July 2018

Proteomic analysis of S-nitrosylated nuclear proteins in rat cortical neurons.

Sci Signal 2018 07 3;11(537). Epub 2018 Jul 3.

Medical Research Council Laboratory for Molecular Cell Biology, University College London, WC1E 6BT London, UK.

Neurons modulate gene expression in response to extrinsic signals to enable brain development, cognition, and learning and to process stimuli that regulate systemic physiological functions. This signal-to-gene communication is facilitated by posttranslational modifications such as S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, but few other nuclear targets of S-nitrosylation have been identified to date. We used S-nitrosothiol resin-assisted capture on NO donor-treated nuclear extracts from rat cortical neurons and identified 614 S-nitrosylated nuclear proteins. Of these, 131 proteins have not previously been shown to be S-nitrosylated in any system, and 555 are previously unidentified targets of S-nitrosylation in neurons. The sites of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines were found at 33% of these sites. In addition, lysine motifs were necessary for promoting the S-nitrosylation of HDAC2 and methyl-CpG binding protein 3 (MBD3). Moreover, S-nitrosylation of the histone-binding protein RBBP7 was necessary for dendritogenesis of cortical neurons in culture. Together, our findings characterize S-nitrosylated nuclear proteins in neurons and identify S-nitrosylation motifs that may be shared with other targets of NO signaling.
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http://dx.doi.org/10.1126/scisignal.aar3396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726469PMC
July 2018

The receptor protein tyrosine phosphatase PTPRJ negatively modulates the CD98hc oncoprotein in lung cancer cells.

Oncotarget 2018 May 4;9(34):23334-23348. Epub 2018 May 4.

Dipartimento di Medicina Sperimentale e Clinica, University Magna Græcia, Campus "S. Venuta", Catanzaro, Italy.

PTPRJ, a receptor protein tyrosine phosphatase strongly downregulated in human cancer, displays tumor suppressor activity by negatively modulating several proteins involved in proliferating signals. Here, through a proteomic-based approach, we identified a list of potential PTPRJ-interacting proteins and among them we focused on CD98hc, a type II glycosylated integral membrane protein encoded by , corresponding to the heavy chain of a heterodimeric transmembrane amino-acid transporter, including LAT1. CD98hc is widely overexpressed in several types of cancers and contributes to the process of tumorigenesis by interfering with cell proliferation, adhesion, and migration. We first validated PTPRJ-CD98hc interaction, then demonstrated that PTPRJ overexpression dramatically reduces CD98hc protein levels in A549 lung cancer cells. In addition, following to the treatment of PTPRJ-transduced cells with MG132, a proteasome inhibitor, CD98hc levels did not decrease compared to controls, indicating that PTPRJ is involved in the regulation of CD98hc proteasomal degradation. Moreover, PTPRJ overexpression combined with CD98hc silencing consistently reduced cell proliferation and triggered apoptosis of lung cancer cells. Interestingly, by interrogating the database, we observed an inverse correlation between and gene expression. Indeed, the non-small cell lung cancers (NSCLCs) of patients showing a short survival rate express the lowest and the highest levels of and , respectively. Therefore, the results reported here contribute to shed lights on PTPRJ signaling in cancer cells: moreover, our findings also support the development of a novel anticancer therapeutic approach by targeting the pathway of PTPRJ that is usually downregulated in highly malignant human neoplasias.
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http://dx.doi.org/10.18632/oncotarget.25101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955124PMC
May 2018

Secretome Analysis of Hypoxia-Induced 3T3-L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity.

Proteomics 2018 04;18(7):e1700260

Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte-secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3-L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin-1 and -2, and matrix metalloproteinase-11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3-L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.
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http://dx.doi.org/10.1002/pmic.201700260DOI Listing
April 2018

Evidence of Altered Glycosylation of Serum Proteins Prior to Pancreatic Cancer Diagnosis.

Int J Mol Sci 2017 Dec 9;18(12). Epub 2017 Dec 9.

Institute for Women's Health, University College London, Gower Street, London WC1E 6BT, UK.

Biomarkers for the early detection of pancreatic cancer are urgently needed. The aim of this pilot study was to evaluate changes in serum -glycoproteins and their glycosylation status prior to clinical presentation of pancreatic cancer that may be potential biomarkers. Prediagnosis serum samples pooled according to five time-to-diagnosis groups and a non-cancer control pool were digested with trypsin, labelled with mass tags, and subjected to titanium dioxide capture, deglycosylation, and 2D-LC-MS/MS profiling. Unbound peptides were profiled in parallel. Across the sample groups, 703 proteins were quantified and 426 putative sites of -glycosylation were identified with evidence of several novel sites. Altered proteins with biomarker potential were predominantly abundant inflammatory response, coagulation, and immune-related proteins. Whilst glycopeptide profiles largely paralleled those of their parent proteins, there was evidence of altered -glycosylation site occupancy or sialic acid content prior to diagnosis for some proteins, most notably of immunoglobulin gamma chains. α-1-Antitrypsin was tested as a biomarker, but found not to complement carbohydrate antigen 19-9 (CA19-9) in early detection of cancer. In conclusion, we provide preliminary evidence of altered glycosylation of several serum proteins prior to pancreatic cancer diagnosis, warranting further investigation of these proteins as early biomarkers. These changes may be largely driven by inflammatory processes that occur in response to tumour formation and progression.
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http://dx.doi.org/10.3390/ijms18122670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751272PMC
December 2017

-Acylbenzenesulfonamide Dihydro-1,3,4-oxadiazole Hybrids: Seeking Selectivity toward Carbonic Anhydrase Isoforms.

ACS Med Chem Lett 2017 Aug 21;8(8):792-796. Epub 2017 Jun 21.

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy.

A series of -acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids () was designed and synthesized with the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumor associated hCA XII. Moreover, resolution of racemic mixture led to the isolation of the levorotatory eutomer exhibiting an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine activity and selectivity toward hCA isozymes. These results can provide further indication for the design and optimization of selective hCA inhibitors.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554910PMC
August 2017

Is mealworm or shrimp allergy indicative for food allergy to insects?

Mol Nutr Food Res 2017 09 18;61(9). Epub 2017 Jul 18.

Department of Dermatology/Allergology, University Medical Center Utrecht, Utrecht, the Netherlands.

Scope: The growing world population is a key driver for the exploration of sustainable protein sources to ensure food security. Mealworm and other insects are promising candidates. Previously we found that shrimp allergic patients are at risk for mealworm allergy, and that mealworm can induce a primary allergy . This study set out to investigate the allergenic potential of edible insects, suggested for human consumption by agencies such as WHO/FAO, in both the shrimp (potentially cross-reactive) and primary mealworm allergic population. The following insects were studied: mealworm, house cricket, giant mealworm, lesser mealworm, African grasshopper, large wax moth, and black soldier fly.

Methods And Results: Fifteen shrimp (mealworm sensitized or allergic) patients and four primary mealworm allergic subjects, who participated in previous studies, were included. All shrimp allergic patients were sensitized to multiple insects with similar response profiles for all insects tested. Primary mealworm allergic patients, showed IgE binding to proteins from only a few insects on immunoblot, although basophil activation test was positive for all tested insects.

Conclusion: Shrimp allergic patients are most likely at risk of food allergy to mealworm and other insects. Primary mealworm allergy does not mean subjects are likely to react to all insects.
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http://dx.doi.org/10.1002/mnfr.201601061DOI Listing
September 2017

Proteomic analysis of protein purified derivative of Mycobacterium bovis.

J Transl Med 2017 04 3;15(1):68. Epub 2017 Apr 3.

Dipartimento di Biologia, Università di Napoli Federico II, Naples, Italy.

Background: Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB).

Methods: Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC-MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system.

Results: Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920.

Conclusions: This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.
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http://dx.doi.org/10.1186/s12967-017-1172-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376687PMC
April 2017

Corrigendum to 'specific changes in the proteomic pattern produced by the BRCA1-Ser1841Asn missense mutation' [International Journal of Biochemistry and Cell Biology (2007) 220-226].

Int J Biochem Cell Biol 2017 07 25;88:236-237. Epub 2017 Mar 25.

Dipartimento di Medicina Sperimentale e Clinica "G. Salvatore", Universit'a degli Studi di Catanzaro "Magna Græcia", Viale Europa Campus Universitario Germaneto, 88100 Catanzaro, Italy.

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http://dx.doi.org/10.1016/j.biocel.2017.03.006DOI Listing
July 2017

Primary respiratory and food allergy to mealworm.

J Allergy Clin Immunol 2017 08 6;140(2):600-603.e7. Epub 2017 Mar 6.

Department of Dermatology and Allergology, University Medical Center Utrecht, Utrecht, The Netherlands; TNO, Zeist, The Netherlands; Utrecht Centre for Food Allergy, Utrecht, The Netherlands.

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http://dx.doi.org/10.1016/j.jaci.2017.01.035DOI Listing
August 2017

An optimized procedure for on-tissue localized protein digestion and quantification using hydrogel discs and isobaric mass tags: analysis of cardiac myxoma.

Anal Bioanal Chem 2017 Apr 11;409(11):2919-2930. Epub 2017 Feb 11.

Research Center for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Campus "S. Venuta", Viale Europa, Loc. Germaneto, 88100, Catanzaro, Italy.

An optimized workflow for multiplexed and spatially localized on-tissue quantitative protein analysis is here presented. The method is based on the use of an enzyme delivery platform, a polymeric hydrogel disc, allowing for a localized digestion directly onto the tissue surface coupled with an isobaric mass tag strategy for peptide labeling and relative quantification. The digestion occurs within such hydrogels, followed by peptide solvent extraction and identification by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Since this is a histology-directed on-tissue analysis, multiple hydrogels were placed onto morphologically and spatially different regions of interest (ROIs) within the tissue surface, e.g., cardiac myxoma tumor vascularized region and the adjacent hypocellular area. After a microwave digestion step (2 min), enzymatically cleaved peptides were labeled using TMT reagents with isobaric mass tags, enabling analysis of multiple samples per experiment. Thus, N = 8 hydrogel-digested samples from cardiac myxoma serial tissue sections (N = 4 from the vascularized ROIs and N = 4 from the adjacent hypocellular areas) were processed and then combined before a single LC-MS/MS analysis. Regulated proteins from both cardiac myxoma regions were assayed in a single experiment. Graphical abstract The workflow for histology-guided on-tissue localized protein digestion followed by isobaric mass tagging and LC-MS/MS analysis for proteins quantification is here summarized.
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http://dx.doi.org/10.1007/s00216-017-0237-6DOI Listing
April 2017

Proteome Speciation by Mass Spectrometry: Characterization of Composite Protein Mixtures in Milk Replacers.

Anal Chem 2016 12 11;88(23):11568-11574. Epub 2016 Nov 11.

Department of Health Sciences, Magna Græcia University of Catanzaro , 88100 Catanzaro, Italy.

The ability of tandem mass spectrometry to determine the primary structure of proteolytic peptides can be exploited to trace back the organisms from which the corresponding proteins were extracted. This information can be important when food products, such as protein powders, can be supplemented with lower-quality starting materials. In order to dissect the origin of proteinaceous material composing a given unknown mixture, a two-step database search strategy for bottom-up nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) data was implemented. A single nanoLC-MS/MS analysis was sufficient not only to determine the qualitative composition of the mixtures under examination, but also to assess the relative percent composition of the various proteomes, if dedicated calibration curves were previously generated. The approach of two-step database search for qualitative analysis and proteome total ion current (pTIC) calculation for quantitative analysis was applied to several binary and ternary mixtures which mimic the composition of milk replacers typically used in calf feeding.
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http://dx.doi.org/10.1021/acs.analchem.6b02848DOI Listing
December 2016

Temperature-dependent regulation of the Ochrobactrum anthropi proteome.

Proteomics 2016 12;16(23):3019-3024

Institute of Microbiology, Department of Health Sciences, "Magna Graecia" University of Catanzaro, Catanzaro, Italy.

Ochrobactrum anthropi is a Gram-negative rod belonging to the Brucellaceae family, able to colonize a variety of environments, and actually reported as a human opportunistic pathogen. Despite its low virulence, the bacterium causes a growing number of hospital-acquired infections mainly, but not exclusively, in immunocompromised patients. The aim of this study was to obtain an overview of the global proteome changes occurring in O. anthropi in response to different growth temperatures, in order to achieve a major understanding of the mechanisms by which the bacterium adapts to different habitats and to identify some potential virulence factors. Combined quantitative mass spectrometry-based proteomics and bioinformatics approaches were carried out on two O. anthropi strains grown at temperatures miming soil/plants habitat (25°C) and human host environment (37°C), respectively. Proteomic analysis led to the identification of over 150 differentially expressed proteins in both strains, out of over 1200 total protein identifications. Among them, proteins responsible for heat shock response (DnaK, GrpE), motility (FliC, FlgG, FlgE), and putative virulence factors (TolB) were identified. The study represents the first quantitative proteomic analysis of O. anthropi performed by high-resolution quantitative mass spectrometry.
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http://dx.doi.org/10.1002/pmic.201600048DOI Listing
December 2016

Effect of thermal processing on mealworm allergenicity.

Mol Nutr Food Res 2015 Sep 1;59(9):1855-64. Epub 2015 Jul 1.

Department of Dermatology/Allergology, University Medical Centre Utrecht (UMCU), Utrecht, The Netherlands.

Scope: The growing world population requires the exploration of new sustainable protein sources to ensure food security. Insects such as mealworm are promising candidates. For safety reasons, a risk assessment, including allergy risks, is needed. Since allergenicity can be influenced by thermal processing, it is highly important to take this into account.

Methods And Results: Fresh mealworm was heat processed and extracted by a sequential extraction method using in succession Tris, urea, and a combined SDS/DTT buffer. Extracts were tested using immunoblot, basophil activation test and skin prick test in 15 shrimp allergic patients, previously indicated as population at risk for mealworm allergy. Immunoblots showed a difference in IgE binding between processed and unprocessed mealworm extracts. However, this was due to change in solubility. Some allergens were soluble in urea buffer, but became more soluble in Tris buffer and vice versa. IgE binding was seen for all extracts in blot and basophil activation test. The results from 13 skin prick tests showed a skin reaction similar between processed and unprocessed mealworm.

Conclusion: Thermal processing did not lower allergenicity but clearly changed solubility of mealworm allergens. A sequential extraction method allowed for assessment of a broader protein panel.
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http://dx.doi.org/10.1002/mnfr.201500138DOI Listing
September 2015
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