Publications by authors named "Giuseppe Grasso"

68 Publications

The interplay between lipid and Aβ amyloid homeostasis in Alzheimer's Disease: risk factors and therapeutic opportunities.

Chem Phys Lipids 2021 05 4;236:105072. Epub 2021 Mar 4.

Consiglio Nazionale delle Ricerche, Istituto di Cristallografia, Catania, Italy. Electronic address:

Alzheimer's Diseases (AD) is characterized by the accumulation of amyloid deposits of Aβ peptide in the brain. Besides genetic background, the presence of other diseases and an unhealthy lifestyle are known risk factors for AD development. Albeit accumulating clinical evidence suggests that an impaired lipid metabolism is related to Aβ deposition, mechanistic insights on the link between amyloid fibril formation/clearance and aberrant lipid interactions are still unavailable. Recently, many studies have described the key role played by membrane bound Aβ assemblies in neurotoxicity. Moreover, it has been suggested that a derangement of the ubiquitin proteasome pathway and autophagy is significantly correlated with toxic Aβ aggregation and dysregulation of lipid levels. Thus, studies focusing on the role played by lipids in Aβ aggregation and proteostasis could represent a promising area of investigation for the design of valuable treatments. In this review we examine current knowledge concerning the effects of lipids in Aβ aggregation and degradation processes, focusing on the therapeutic opportunities that a comprehensive understanding of all biophysical, biochemical, and biological processes involved may disclose.
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http://dx.doi.org/10.1016/j.chemphyslip.2021.105072DOI Listing
May 2021

The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism.

J Enzyme Inhib Med Chem 2021 Dec;36(1):183-187

Department of Chemical Sciences, University of Catania, Catania, Italy.

We have applied a recently developed HPLC-MS enzymatic assay to investigate the cryptic peptides generated by the action of the insulin-degrading enzyme (IDE) on some neuropeptides (NPs) involved in the development of tolerance and dependence to opioids. Particularly, the tested NPs are generated from the NPFF precursor (pro-NPFF (A)): NPFF (FLFQPQRF) and NPAF (AGEGLSSPFWSLAAPQRF). The results show that IDE is able to cleave NPFF and NPAF, generating specific cryptic peptides. As IDE is also responsible for the processing of many other peptides in the brain (amyloid beta protein among the others), we have also performed competitive degradation assays using mixtures of insulin and the above mentioned NPs. Data show that insulin is able to slow down the degradation of both NPs tested, whereas, surprisingly, NPAF is able to accelerate insulin degradation, hinting IDE as the possible link responsible of the mutual influence between insulin and NPs metabolism.
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http://dx.doi.org/10.1080/14756366.2020.1850712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801110PMC
December 2021

Histological and Histomorphometric Evaluation of New Bone Formation after Maxillary Sinus Augmentation with Two Different Osteoconductive Materials: A Randomized, Parallel, Double-Blind Clinical Trial.

Materials (Basel) 2020 Dec 3;13(23). Epub 2020 Dec 3.

Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

This study aimed to investigate the histological features of deproteinized equine bone mineral (DEBM) and anorganic bovine bone (ABB) after human sinus augmentation with the lateral approach. Twenty-three sinus augmentations were performed in 16 patients (male: 10/female: 6) using DEBM or ABB in a randomized fashion. Healing took place over the next 6 months. Bone core biopsies (N = 23) were obtained for each subject prior to placing the dental implants. The biopsies were processed for both histological descriptions and histomorphometric analysis. Statistical analyses were applied as appropriate, defining statistical significance as < 0.05. Core bone biopsies revealed no differences in terms of newly formed bone between groups, or differences in terms of tissue inflammation. Both DEBM and ABB appear to be suitable biomaterials for bone augmentation in sinus lift surgery in the short term. However, dedicated studies are required to confirm these results and their stability in the long term.
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http://dx.doi.org/10.3390/ma13235520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729968PMC
December 2020

Defective proteasome biogenesis into skin fibroblasts isolated from Rett syndrome subjects with MeCP2 non-sense mutations.

Biochim Biophys Acta Mol Basis Dis 2020 07 8;1866(7):165793. Epub 2020 Apr 8.

Dept of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy. Electronic address:

Rett Syndrome (RTT) is a rare X-linked neurodevelopmental disorder which affects about 1: 10000 live births. In >95% of subjects RTT is caused by a mutation in Methyl-CpG binding protein-2 (MECP2) gene, which encodes for a transcription regulator with pleiotropic genetic/epigenetic activities. The molecular mechanisms underscoring the phenotypic alteration of RTT are largely unknown and this has impaired the development of therapeutic approaches to alleviate signs and symptoms during disease progression. A defective proteasome biogenesis into two skin primary fibroblasts isolated from RTT subjects harbouring non-sense (early-truncating) MeCP2 mutations (i.e., R190fs and R255X) is herewith reported. Proteasome is the proteolytic machinery of Ubiquitin Proteasome System (UPS), a pathway of overwhelming relevance for post-mitotic cells metabolism. Molecular, transcription and proteomic analyses indicate that MeCP2 mutations down-regulate the expression of one proteasome subunit, α7, and of two chaperones, PAC1 and PAC2, which bind each other in the earliest step of proteasome biogenesis. Furthermore, this molecular alteration recapitulates in neuron-like SH-SY5Y cells upon silencing of MeCP2 expression, envisaging a general significance of this transcription regulator in proteasome biogenesis.
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http://dx.doi.org/10.1016/j.bbadis.2020.165793DOI Listing
July 2020

THE USE OF MASS SPECTROMETRY TO STUDY ZN-METALLOPROTEASE-SUBSTRATE INTERACTIONS.

Authors:
Giuseppe Grasso

Mass Spectrom Rev 2020 09 3;39(5-6):574-585. Epub 2020 Jan 3.

Department of Chemical Sciences, Università degli Studi di Catania, Viale Andrea Doria 6, Catania, 95125, Italy.

Zinc metalloproteases (ZnMPs) participate in diverse biological reactions, encompassing the synthesis and degradation of all the major metabolites in living organisms. In particular, ZnMPs have been recognized to play a very important role in controlling the concentration level of several peptides and/or proteins whose homeostasis has to be finely regulated for the correct physiology of cells. Dyshomeostasis of aggregation-prone proteins causes pathological conditions and the development of several different diseases. For this reason, in recent years, many analytical approaches have been applied for studying the interaction between ZnMPs and their substrates and how environmental factors can affect enzyme activities. In this scenario, mass spectrometric methods occupy a very important role in elucidating different aspects of ZnMPs-substrates interaction. These range from identification of cleavage sites to quantitation of kinetic parameters. In this work, an overview of all the main achievements regarding the application of mass spectrometric methods to investigating ZnMPs-substrates interactions is presented. A general experimental protocol is also described which may prove useful to the study of similar interactions. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.
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http://dx.doi.org/10.1002/mas.21621DOI Listing
September 2020

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from β-Amyloid Toxicity.

ChemMedChem 2020 02 17;15(3):302-316. Epub 2019 Dec 17.

Consiglio Nazionale delle Ricerche, Istituto di Cristallografia, Via P. Gaifami 18, 95126, Catania, Italy.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from β-amyloid (Aβ) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aβ degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.
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http://dx.doi.org/10.1002/cmdc.201900612DOI Listing
February 2020

The miR-205-5p/BRCA1/RAD17 Axis Promotes Genomic Instability in Head and Neck Squamous Cell Carcinomas.

Cancers (Basel) 2019 09 11;11(9). Epub 2019 Sep 11.

Oncogenomic and Epigenetic Unit, Department of Diagnostic Research and Technological Innovation, IRCCS Regina Elena National Cancer Institute, 00144 Rome, Italy.

Defective DNA damage response (DDR) is frequently associated with tumorigenesis. Abrogation of DDR leads to genomic instability, which is one of the most common characteristics of human cancers. mutations with gain-of-function activity are associated with tumors under high replicative stress, high genomic instability, and reduced patient survival. The and genes encode two pivotal DNA repair proteins required for proper cell-cycle regulation and maintenance of genomic stability. We initially evaluated whether miR-205-5p, a microRNA (miRNA) highly expressed in head and neck squamous cell carcinoma (HNSCC), targeted BRCA1 and RAD17 expression. We found that, in vitro and in vivo, and are targets of miR-205-5p in HNSCC, leading to inefficient DNA repair and increased chromosomal instability. Conversely, miR-205-5p downregulation increased BRCA1 and RAD17 messenger RNA (mRNA) levels, leading to a reduction in in vivo tumor growth. Interestingly, miR-205-5p expression was significantly anti-correlated with and targets. Furthermore, we documented that miR-205-5p expression was higher in tumoral and peritumoral HNSCC tissues than non-tumoral tissues in patients exhibiting reduced local recurrence-free survival. Collectively, these findings unveil miR-205-5p's notable role in determining genomic instability in HNSCC through its selective targeting of BRCA1 and RAD17 gene expression. High miR-205-5p levels in the peritumoral tissues might be relevant for the early detection of minimal residual disease and pre-cancer molecular alterations involved in tumor development.
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http://dx.doi.org/10.3390/cancers11091347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771082PMC
September 2019

IDE Degrades Nociceptin/Orphanin FQ through an Insulin Regulated Mechanism.

Int J Mol Sci 2019 Sep 10;20(18). Epub 2019 Sep 10.

Department of Chemical Sciences, University of Catania, 95125 Catania, Italy.

Insulin-degrading enzyme (IDE) was applied to catalyze hydrolysis of Nociceptin/Orphanin 1-16 (OFQ/N) to show the involvement of the enzyme in degradation of neuropeptides engaged in pain transmission. Moreover, IDE degradative action towards insulin (Ins) was inhibited by the OFQ/N fragments, suggesting a possible regulatory mechanism in the central nervous system. It has been found that OFQ/N and Ins affect each other degradation by IDE, although in a different manner. Indeed, while the digestion of OFQ/N is significantly affected by the presence of Ins, the kinetic profile of the Ins hydrolysis is not affected by the presence of OFQ/N. However, the main hydrolytic fragments of OFQ/N produced by IDE exert inhibitory activity towards the IDE-mediated Ins degradation. Here, we present the results indicating that, besides Ins, IDE cleaves neuropeptides and their released fragments act as inhibitors of IDE activity toward Ins. Having in mind that IDE is present in the brain, which also contains Ins receptors, it cannot be excluded that this enzyme indirectly participates in neural communication of pain signals and that neuropeptides involved in pain transmission may contribute to the regulation of IDE activity. Finally, preliminary results on the metabolism of OFQ/N, carried out in the rat spinal cord homogenate in the presence of various inhibitors specific for different classes of proteases, show that OFQ/N proteolysis in rat spinal cord could be due, besides IDE, also to a cysteine protease not yet identified.
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http://dx.doi.org/10.3390/ijms20184447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770469PMC
September 2019

Dropwort-induced metabolic reprogramming restrains YAP/TAZ/TEAD oncogenic axis in mesothelioma.

J Exp Clin Cancer Res 2019 Aug 9;38(1):349. Epub 2019 Aug 9.

Oncogenomic and Epigenetic Unit, Department of Research, Diagnosis and Innovative Technologies, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi, 53, 00144, Rome, Italy.

Background: Over the past decade, newly designed cancer therapies have not significantly improved the survival of patients diagnosed with Malignant Pleural Mesothelioma (MPM). Among a limited number of genes that are frequently mutated in MPM several of them encode proteins that belong to the HIPPO tumor suppressor pathway.

Methods: The anticancer effects of the top flower standardized extract of Filipendula vulgaris (Dropwort) were characterized in "in vitro" and "in vivo" models of MPM. At the molecular level, two "omic" approaches were used to investigate Dropwort anticancer mechanism of action: a metabolomic profiling and a phosphoarray analysis.

Results: We found that Dropwort significantly reduced cell proliferation, viability, migration and in vivo tumor growth of MPM cell lines. Notably, Dropwort affected viability of tumor-initiating MPM cells and synergized with Cisplatin and Pemetrexed in vitro. Metabolomic profiling revealed that Dropwort treatment affected both glycolysis/tricarboxylic acid cycle as for the decreased consumption of glucose, pyruvate, succinate and acetate, and the lipid metabolism. We also document that Dropwort exerted its anticancer effects, at least partially, promoting YAP and TAZ protein ubiquitination.

Conclusions: Our findings reveal that Dropwort is a promising source of natural compound(s) for targeting the HIPPO pathway with chemo-preventive and anticancer implications for MPM management.
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http://dx.doi.org/10.1186/s13046-019-1352-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689183PMC
August 2019

Site directed mutagenesis of insulin-degrading enzyme allows singling out the molecular basis of peptidase versus E1-like activity: the role of metal ions.

Metallomics 2019 02;11(2):278-281

Institute of Biostructures and Bioimaging, National Research Council, Catania, Italy.

Four specifically designed IDE mutants have been used to unveil the molecular basis of peptidase versus E1-like activity of the enzyme. We have found that physiological concentrations of copper(ii) ions inhibit the proteolytic activity of the enzyme towards small and large substrates but have no effect on the E1-like activity of the enzyme.
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http://dx.doi.org/10.1039/c8mt00288fDOI Listing
February 2019

Commentary on Ivancic et al.: Enzyme kinetics from circular dichroism of insulin reveals mechanistic insights into the regulation of insulin-degrading enzyme.

Authors:
Giuseppe Grasso

Biosci Rep 2018 12 28;38(6). Epub 2018 Nov 28.

Department of Chemical Sciences, University of Catania, Viale Andrea Doria 6, 95125, Catania, Italy

Despite the enormous number of therapeutic advances in medicine, nowadays many diseases are still incurable, mainly due to the lack of knowledge of the pathological biochemical pathways triggering those diseases. For this reason, it is compulsory for the scientific community to investigate and unveil the biomolecular mechanisms responsible for the development of those diseases, such as Alzheimer's disease and diabetes, which are widespread all over the world. In this scenario, it is of paramount importance to develop new analytical techniques and experimental procedures that are capable to make the above-mentioned investigations feasible. These new methods should allow easy performable analysis carried out in a label-free environment, in order to give reliable answers to specific biochemical questions. A recent paper published on by Ivancic et al. (https://doi.org/10.1042/BSR20181416) proposes a new analytical technique capable to reveal some mechanistic insights into the regulation of insulin-degrading enzyme (IDE), a protein involved in the above-mentioned diseases. IDE is a multifaceted enzyme having different and not well-defined roles in the cell, but it is primarily a proteolytic enzyme capable to degrade several different amyloidogenic substrates involved in different diseases. Moreover, many molecules are responsible for IDE activity modulation so that understanding how IDE activity is regulated represents a very challenging analytical task. The new analytical approach proposed by Ivancic et al. reports on the possibility to study IDE activity in an unbiased and label-free manner, representing a valid alternative assay for the investigation of any proteases degradative activity.
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http://dx.doi.org/10.1042/BSR20181555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6259020PMC
December 2018

Mass spectrometry is a multifaceted weapon to be used in the battle against Alzheimer's disease: Amyloid beta peptides and beyond.

Authors:
Giuseppe Grasso

Mass Spectrom Rev 2019 01 15;38(1):34-48. Epub 2018 Jun 15.

Department of Chemical Sciences, University of Catania, Catania, Italy.

Amyloid-β peptide (Aβ) accumulation and aggregation have been considered for many years the main cause of Alzheimer's disease (AD), and therefore have been the principal target of investigation as well as of the proposed therapeutic approaches (Grasso [2011] Mass Spectrom Rev. 30: 347-365). However, the amyloid cascade hypothesis, which considers Aβ accumulation the only causative agent of the disease, has proven to be incomplete if not wrong. In recent years, actors such as metal ions, oxidative stress, and other cofactors have been proposed as possible co-agents or, in some cases, main causative factors of AD. In this scenario, MS investigation has proven to be fundamental to design possible diagnostic strategies of this elusive disease, as well as to understand the biomolecular mechanisms involved, in the attempt to find a possible therapeutic solution. We review the current applications of MS in the search for possible Aβ biomarkers of AD to help the diagnosis of the disease. Recent examples of the important contributions that MS has given to prove or build theories on the molecular pathways involved with such terrible disease are also reviewed.
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http://dx.doi.org/10.1002/mas.21566DOI Listing
January 2019

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S.

Cell Mol Life Sci 2018 Sep 28;75(18):3441-3456. Epub 2018 Mar 28.

Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Via Montpellier 1, 00133, Rome, Italy.

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE) ≤ 30 nM and activating at (IDE) ≥ 30 nM. Only an activating effect is observed in a yeast mutant locked in the "open" conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S "open"-"closed" allosteric equilibrium. Protein-protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S.
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http://dx.doi.org/10.1007/s00018-018-2807-yDOI Listing
September 2018

Carnosine protects pancreatic beta cells and islets against oxidative stress damage.

Mol Cell Endocrinol 2018 10 26;474:105-118. Epub 2018 Feb 26.

Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad alta specializzazione), Palermo, Italy.

Islet transplantation is a valid therapeutic option for type 1 diabetes treatment. However, in this procedure one of the major problems is the oxidative stress produced during pancreatic islet isolation. The aim of our study was to evaluate potential protective effects of L-carnosine and its isomer D-carnosine against oxidative stress. We evaluated the carnosine effect on cell growth, cell death, insulin production, and the main markers of oxidative stress in rat and murine stressed beta cell lines as well as in human pancreatic islets. Both isomers clearly inhibited hydrogen peroxide induced cytotoxicity, with a decrease in intracellular reactive oxygen and nitrogen species, prevented hydrogen peroxide induced apoptosis/necrosis, nitrite production, and reduced glucose-induced insulin secretion. In addition, NF-κB expression/translocation and nitrated protein induced in stressed cells was significantly reduced. Furthermore, both isomers improved survival and function, and decreased reactive oxygen and nitrogen species, and nitrite and nitrotyrosine production in human islets cultured for 1, 3, and 7 days. These results seem to indicate that both L and D-carnosine have a significant cytoprotective effect by reducing oxidative stress in beta cell lines and human islets, suggesting their potential use to improve islet survival during the islet transplantation procedure.
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http://dx.doi.org/10.1016/j.mce.2018.02.016DOI Listing
October 2018

Synthesis and Antibacterial Activity of Polymerizable Acryloyloxyalkyltriethyl Ammonium Salts.

Chempluschem 2017 Oct 27;82(10):1235-1244. Epub 2017 Jun 27.

Department of Chemistry and Chemical Technologies, University of Calabria, Via P. Bucci, 12/C, 87036, Arcavacata di Rende, CS, Italy.

This study reports an efficient and practical synthetic approach for the synthesis of a particularly important class of polymerizable quaternary ammonium salts (PQASs), that is, acryloxyalkyltriethylammonium bromides (AATEABs), which may find application as antimicrobial coatings for commercial membranes with antifouling and anti-biofouling properties, to be used for wastewater treatment. The synthetic method is based on a simple two-step procedure from commercially available substrates, entirely carried out under air and without any need for chromatographic purification. All the newly synthesized AATEABs were tested for their antimicrobial activity, and the results showed that AATEABs bearing an alkyl chain of 11 and particularly 12 carbon atoms possessed significant activity against Gram positive bacteria and yeast strains.
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http://dx.doi.org/10.1002/cplu.201700194DOI Listing
October 2017

Synthesis and Antibacterial Activity of Polymerizable Acryloyloxyalkyltriethyl Ammonium Salts.

Chempluschem 2017 Oct 9;82(10):1233-1234. Epub 2017 Aug 9.

Department of Chemistry and Chemical Technologies, University of Calabria, Via P. Bucci, 12/C, 87036, Arcavacata di Rende, CS, Italy.

Invited for this month's cover are the collaborating groups at the University of Calabria (Unical, Italy)-Department of Chemistry and Chemical Technologies (CTC) and Department of Pharmacy and Health and Nutritional Sciences (PHNS), at the National Research Council (CNR, Italy), Institute on Membrane Technology (ITM), and at the Karlsruhe University of Applied Sciences (HSKA, Germany), Institute of Applied Research (IAR), which cooperated in the framework of the European Union's Horizon 2020 project "VicInAqua". The front cover shows the structure of an acryloxyalkyltrithylammonium (AATEA) cation, characterized by a polymerizable end, a long-chain alkyl linker, and a quaternary ammonium moiety. The bromide salts (AATEABs) have been synthesized by a two-step procedure carried out entirely under air without any need for chromatographic purifications. Some AATEABs have shown significant antimicrobial activity and represent useful precursors for the preparation of polymeric films with antimicrobial properties. Read the full text of the article at 10.1002/cplu.201700194.
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http://dx.doi.org/10.1002/cplu.201700329DOI Listing
October 2017

Surface tailoring of polyacrylate-grafted graphene oxide for controlled interactions at the biointerface.

J Colloid Interface Sci 2017 Nov 21;506:532-542. Epub 2017 Jul 21.

Department of Chemical Sciences, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy. Electronic address:

The actual surface termination and lateral size of a nanomaterial is crucial in its interaction with biomolecules at the aqueous interface. Graphene oxide (GO) nanosheets have been demonstrated as promising nanoplatform for both diagnostic and therapeutic applications. To this respect, 'smart' GO nanocarriers have been obtained by the surface functionalisation with polymers sensitive, e.g., to pH, as the polyacrylate (PAA) case. In this work, hybrid GO/PAA samples prepared respectively at low (GOPAA) or high (GOPAA) monomer grafting ratio, were scrutinised both theoretically, by molecular dynamic calculations, and experimentally by a multitechnique approach, including spectroscopic (UV-visible, fluorescence, Raman, Attenuated-total reflectance-Fourier transformed infrared and X-ray photoelectron spectroscopies), spectrometric (time-of-flight secondary ion and electrospray ionisation mass spectrometries) and microscopic (atomic force and confocal microscopies) methods. The actual surface termination, evaluated in terms of the relative ratio between polar and dispersive groups at the surface of the GO/polymer systems, was found to correlate with the average orientation of hydrophilic/hydrophobic domains of albumin, used as model protein. Moreover, the comparison among GO, GO-PAA and GO-PAA in the optical response at the interface with aqueous solutions, both at acid and at physiological pH, showed that the hybrid GO-polymer platform could be suitable not only to exploit a pH-triggered drug release but also for a modulation of the GO intrinsic emission properties. Energy transfer experiments on the GO/polymer oxide/fluorescein-labelled albumin/doxorubicin assembly showed significant differences for GO and GO-PAA samples, thus demonstrating the occurrence of different electronic processes at the hybrid nano-bio-interfaces. Confocal microscopy studies of cellular uptake in neuroblastoma cells confirmed the promising potentialities of the developed nanoplatform for applications at the biointerface.
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http://dx.doi.org/10.1016/j.jcis.2017.07.080DOI Listing
November 2017

Multiple functions of insulin-degrading enzyme: a metabolic crosslight?

Crit Rev Biochem Mol Biol 2017 10 21;52(5):554-582. Epub 2017 Jun 21.

a Department of Clinical Sciences and Translation Medicine , University of Roma Tor Vergata , Roma , Italy.

Insulin-degrading enzyme (IDE) is a ubiquitous zinc peptidase of the inverzincin family, which has been initially discovered as the enzyme responsible for insulin catabolism; therefore, its involvement in the onset of diabetes has been largely investigated. However, further studies on IDE unraveled its ability to degrade several other polypeptides, such as β-amyloid, amylin, and glucagon, envisaging the possible implication of IDE dys-regulation in the "aggregopathies" and, in particular, in neurodegenerative diseases. Over the last decade, a novel scenario on IDE biology has emerged, pointing out a multi-functional role of this enzyme in several basic cellular processes. In particular, latest advances indicate that IDE behaves as a heat shock protein and modulates the ubiquitin-proteasome system, suggesting a major implication in proteins turnover and cell homeostasis. In addition, recent observations have highlighted that the regulation of glucose metabolism by IDE is not merely based on its largely proposed role in the degradation of insulin in vivo. There is increasing evidence that improper IDE function, regulation, or trafficking might contribute to the etiology of metabolic diseases. In addition, the enzymatic activity of IDE is affected by metals levels, thus suggesting a role also in the metal homeostasis (metallostasis), which is thought to be tightly linked to the malfunction of the "quality control" machinery of the cell. Focusing on the physiological role of IDE, we will address a comprehensive vision of the very complex scenario in which IDE takes part, outlining its crucial role in interconnecting several relevant cellular processes.
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http://dx.doi.org/10.1080/10409238.2017.1337707DOI Listing
October 2017

Inhibition of Aβ Amyloid Growth and Toxicity by Silybins: The Crucial Role of Stereochemistry.

ACS Chem Neurosci 2017 08 9;8(8):1767-1778. Epub 2017 Jun 9.

Institute of Biostructures and Bioimages-Catania, National Research Council, Via Paolo Gaifami 8, 95126 Catania, Italy.

The self-assembling of the amyloid β (Aβ) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer's disease (AD). Based on the "amyloid hypothesis", many efforts have been devoted to designing molecules able to halt disease progression by inhibiting Aβ self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment MVGGVV of Aβ40. Consequently to silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments LVFF and NKGAII of Aβ40 which shows a high tendency to form bend, turn, and β-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment LVFF but lead to the formation of different small-sized, ThT-positive Aβ aggregates. Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aβ indicated that silybin B is the most effective of the four compounds in counteracting Aβ proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to silybin B as a promising lead compound for further development in anti-AD therapeutics.
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http://dx.doi.org/10.1021/acschemneuro.7b00110DOI Listing
August 2017

AMPK β1 reduces tumor progression and improves survival in p53 null mice.

Mol Oncol 2017 09 28;11(9):1143-1155. Epub 2017 Jun 28.

Department of Medicine, McMaster University, Hamilton, Canada.

The AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK β1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK β1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK β1 accelerates the appearance of a T-cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin-1β (IL1β), reductions in acetyl-CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK β1 subunit accelerate the development of T-cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53-mutant tumors.
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http://dx.doi.org/10.1002/1878-0261.12079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579332PMC
September 2017

Atmospheric pressure MALDI for the noninvasive characterization of carbonaceous ink from Renaissance documents.

Anal Bioanal Chem 2017 Jun 21;409(16):3943-3950. Epub 2017 Apr 21.

Department of Chemistry, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.

The analytical methods that are usually applied to determine the compositions of inks from ancient manuscripts usually focus on inorganic components, as in the case of iron gall ink. In this work, we describe the use of atmospheric pressure/matrix-assisted laser desorption ionization-mass spectrometry (AP/MALDI-MS) as a spatially resolved analytical technique for the study of the organic carbonaceous components of inks used in handwritten parts of ancient books for the first time. Large polycyclic aromatic hydrocarbons (L-PAH) were identified in situ in the ink of XVII century handwritten documents. We prove that it is possible to apply MALDI-MS as a suitable microdestructive diagnostic tool for analyzing samples in air at atmospheric pressure, thus simplifying investigations of the organic components of artistic and archaeological objects. The interpretation of the experimental MS results was supported by independent Raman spectroscopic investigations. Graphical abstract Atmospheric pressure/MALDI mass spectrometry detects in situ polycyclic aromatic hydrocarbons in the carbonaceous ink of XVII century manuscripts.
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http://dx.doi.org/10.1007/s00216-017-0341-7DOI Listing
June 2017

Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin.

J Mass Spectrom 2017 01;52(1):36-42

Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, 19104, USA.

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jms.3897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360464PMC
January 2017

Ubiquitin Associates with the N-Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions.

Chemistry 2016 Dec 19;22(49):17767-17775. Epub 2016 Oct 19.

Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Via P. Gaifami18, 95126, Catania, Italy.

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (K =8.6 10  m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.
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http://dx.doi.org/10.1002/chem.201603650DOI Listing
December 2016

Copper, differently from zinc, affects the conformation, oligomerization state and activity of bradykinin.

Metallomics 2016 08;8(8):750-61

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale Andrea Doria 6, 95125, Catania, Italy.

The sole role of bradykinin (BK) as an inflammatory mediator is controversial, as recent data also support an anti-inflammatory role for BK in Alzheimer's disease (AD). The involvement of two different receptors (B1R and B2R) could be a key to understand this issue. However, although copper and zinc dyshomeostasis has been demonstrated to be largely involved in the development of AD, a detailed study of the interaction of BK with these two metal ions has never been addressed. In this work, we have applied mass spectrometry, circular dichroism as well as computational methods in order to assess if copper and zinc have the ability to modulate the conformation and oligomerization of BK. In addition, we have correlated the chemical data with the effect of metals on the activity of BK analyzed in cell cultures by biochemical procedures. The biochemical analyses on monocyte/macrophage cell culture (THP-1 Cell Line human) in line with the effect of metals on the conformation of BK showed that the presence of copper can affect the signaling cascade mediated by the BK receptors. The results obtained show a further role of metal ions, particularly copper, in the development and outcome of neuroinflammatory diseases. The possible implications in AD are discussed.
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http://dx.doi.org/10.1039/c6mt00067cDOI Listing
August 2016

A neglected modulator of insulin-degrading enzyme activity and conformation: The pH.

Biophys Chem 2015 Aug-Sep;203-204:33-40. Epub 2015 May 27.

Istituto Biostrutture e Bioimmagini, CNR, Via P. Gaifami 18, 95126 Catania, Italy.

Insulin-degrading enzyme (IDE), a ubiquitously expressed zinc metalloprotease, has multiple activities in addition to insulin degradation and its malfunction is believed to connect type 2 diabetes with Alzheimer's disease. IDE has been found in many different cellular compartments, where it may experience significant physio-pathological pH variations. However, the exact role of pH variations on the interplay between enzyme conformations, stability, oligomerization state and catalysis is not understood. Here, we use ESI mass spectrometry, atomic force microscopy, surface plasmon resonance and circular dichroism to investigate the structure-activity relationship of IDE at different pH values. We show that acidic pH affects the ability of the enzyme to bind the substrate and decrease the stability of the protein by inducing an α-helical bundle conformation with a concomitant dissociation of multi-subunit IDE assemblies into monomeric units and loss of activity. These effects suggest a major role played by electrostatic forces in regulating multi-subunit enzyme assembly and function. Our results clearly indicate a pH dependent coupling among enzyme conformation, assembly and stability and suggest that cellular acidosis can have a large effect on IDE oligomerization state, inducing an enzyme inactivation and an altered insulin degradation that could have an impact on insulin signaling.
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http://dx.doi.org/10.1016/j.bpc.2015.05.010DOI Listing
March 2016

Monitoring the biomolecular interactions and the activity of Zn-containing enzymes involved in conformational diseases: experimental methods for therapeutic purposes.

Authors:
Giuseppe Grasso

Adv Protein Chem Struct Biol 2014 26;97:115-42. Epub 2014 Sep 26.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Catania, Italy.

Zinc metalloproteases (ZnMPs) participate in diverse biological reactions, encompassing the synthesis and degradation of all the major metabolites in living organisms. In particular, ZnMPs have been recognized to play a very important role in controlling the concentration level of several peptides and/or proteins whose homeostasis has to be finely regulated for the correct physiology of cells. Dyshomeostasis of aggregation-prone proteins causes pathological conditions and the development of several different diseases. For this reason, in recent years, many analytical approaches have been applied for studying the interaction between ZnMPs and their substrates/inhibitors and how environmental factors can affect enzyme activities. In this scenario, nuclear magnetic resonance, X-ray diffraction, mass spectrometric (MS), and optical methods occupy a very important role in elucidating different aspects of the ZnMPs-substrates/inhibitors interaction, ranging from identification of cleavage sites to quantitation of kinetic parameters and inhibition constants. Here, an overview of all the main achievements in the application of different experimental approaches with special attention to MS methods to the investigation of ZnMPs-substrates/inhibitors interaction is given. A general MS experimental protocol which has been proved to be useful to study such interactions is also described.
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http://dx.doi.org/10.1016/bs.apcsb.2014.08.002DOI Listing
July 2016

Metabolism of cryptic peptides derived from neuropeptide FF precursors: the involvement of insulin-degrading enzyme.

Int J Mol Sci 2014 Sep 22;15(9):16787-99. Epub 2014 Sep 22.

Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland.

The term "cryptome" refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes.
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http://dx.doi.org/10.3390/ijms150916787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200852PMC
September 2014

The role of copper(II) in the aggregation of human amylin.

Metallomics 2014 Oct 1;6(10):1841-52. Epub 2014 Aug 1.

Dottorato Internazionale in Biomedicina Traslazionale, Università degli Studi di Catania, Viale Andrea Doria 6, 95125, Catania, Italy.

Amylin is a 37-residue peptide hormone produced by the islet β-cells of pancreas and the formation of amylin aggregates is strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aβ and α-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported.
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http://dx.doi.org/10.1039/c4mt00130cDOI Listing
October 2014

Neoadjuvant therapy for locally advanced melanoma: new strategies with targeted therapies.

Onco Targets Ther 2014 19;7:1115-21. Epub 2014 Jun 19.

Medical Oncology Department, San Vincenzo Hospital, Taormina, Messina, Italy.

Neoadjuvant chemotherapy has been successfully tested in several bulky solid tumors, but it has not been utilized in advanced cutaneous melanoma, primarily because effective medical treatments for this disease have been lacking. However, with the development of new immunotherapies (monoclonal antibodies specific for cytotoxic T lymphocyte-associated antigen 4 [anti-CTLA-4] and programmed death protein-1 [anti-PD1]) and small molecules interfering with intracellular pathways (anti-BRAF and mitogen-activated protein kinase kinase [anti- MEK]) the use of this approach is becoming a viable treatment strategy for locally advanced melanoma. The neoadjuvant setting provides a double opportunity for a better knowledge of these drugs: a short-term evaluation of their intrinsic activity, and a deeper analysis of their action and resistance-induction mechanisms. BRAF inhibitors seem to be ideal candidates for the neoadjuvant setting, because of their prompt, repeatedly confirmed response in V600E BRAF-mutant metastatic melanoma. In this report we summarize studies focused on the neoadjuvant use of traditional medical treatments in advanced melanoma and anecdotal cases of this approach with the use of biologic therapies. Moreover, we discuss our experience with neoadjuvant targeted therapy as a priming for radical surgery in a patient with BRAF V600E mutation-positive advanced melanoma.
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http://dx.doi.org/10.2147/OTT.S62699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069135PMC
June 2014

Metal complexes and metalloproteases: targeting conformational diseases.

Metallomics 2014 Aug;6(8):1346-57

Chemistry Department, Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, 95125, Catania, Italy.

In recent years many metalloproteases (MPs) have been shown to play important roles in the development of various pathological conditions. Although most of the literature is focused on matrix MPs (MMPs), many other MPs have been demonstrated to be involved in the degradation of peptides or proteins whose accumulation and dyshomeostasis are considered as being responsible for the development of conformational diseases, i.e., diseases where non-native protein conformations lead to protein aggregation. It seems clear that, at least in principle, it must be possible to control the levels of many aggregation-prone proteins not only by reducing their production, but also by enhancing their catabolism. Metal complexes that can perform this function were designed and tested according to at least two different strategies: (i) intervening on the endogenous MPs by directly or indirectly modulating their activity; (ii) acting as artificial MPs, replacing or synergistically functioning with endogenous MPs. These two different bioinorganic approaches are widely represented in the current literature and the aim of this review is to rationally organize and discuss both of them so as to give a critical insight into these approaches and highlighting their limitations and future perspectives.
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http://dx.doi.org/10.1039/c4mt00076eDOI Listing
August 2014
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