Publications by authors named "Paola Turano"

87 Publications

NMR reveals the metabolic changes induced by auranofin in A2780 cancer cells: evidence for glutathione dysregulation.

Dalton Trans 2021 May;50(18):6349-6355

Center of Magnetic Resonance, University of Florence, via Luigi Sacconi 6, 50019, Sesto Fiorentino, Firenze, Italy. and Consorzio Interuniversitario Risonanze Magnetiche di Metallo Proteine (CIRMMP), via Luigi Sacconi 6, 50019, Sesto Fiorentino, Firenze, Italy and Department of Chemistry, University of Florence, via della Lastruccia 3-13, 50019, Sesto Fiorentino, Firenze, Italy.

NMR metabolomics represents a powerful tool to characterize the cellular effects of drugs and gain detailed insight into their mode of action. Here, we have exploited NMR metabolomics to illustrate the changes in the metabolic profile of A2780 ovarian cancer cells elicited by auranofin (AF), a clinically approved gold drug now repurposed as an anticancer agent. An early and large increase in intracellular glutathione is highlighted as the main effect of the treatment accompanied by small but significant changes in the levels of a few additional metabolites; the general implications of these findings are discussed in the frame of the current mechanistic knowledge of AF.
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http://dx.doi.org/10.1039/d1dt00750eDOI Listing
May 2021

Metabolomic/lipidomic profiling of COVID-19 and individual response to tocilizumab.

PLoS Pathog 2021 02 1;17(2):e1009243. Epub 2021 Feb 1.

Magnetic Resonance Center (CERM), University of Florence, Sesto Fiorentino, Florence, Italy.

The current pandemic emergence of novel coronavirus disease (COVID-19) poses a relevant threat to global health. SARS-CoV-2 infection is characterized by a wide range of clinical manifestations, ranging from absence of symptoms to severe forms that need intensive care treatment. Here, plasma-EDTA samples of 30 patients compared with age- and sex-matched controls were analyzed via untargeted nuclear magnetic resonance (NMR)-based metabolomics and lipidomics. With the same approach, the effect of tocilizumab administration was evaluated in a subset of patients. Despite the heterogeneity of the clinical symptoms, COVID-19 patients are characterized by common plasma metabolomic and lipidomic signatures (91.7% and 87.5% accuracy, respectively, when compared to controls). Tocilizumab treatment resulted in at least partial reversion of the metabolic alterations due to SARS-CoV-2 infection. In conclusion, NMR-based metabolomic and lipidomic profiling provides novel insights into the pathophysiological mechanism of human response to SARS-CoV-2 infection and to monitor treatment outcomes.
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http://dx.doi.org/10.1371/journal.ppat.1009243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7877736PMC
February 2021

A geroscience approach for Parkinson's disease: Conceptual framework and design of PROPAG-AGEING project.

Mech Ageing Dev 2021 03 29;194:111426. Epub 2020 Dec 29.

Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine (CIRMMP), Florence, Italy.

Advanced age is the major risk factor for idiopathic Parkinson's disease (PD), but to date the biological relationship between PD and ageing remains elusive. Here we describe the rationale and the design of the H2020 funded project "PROPAG-AGEING", whose aim is to characterize the contribution of the ageing process to PD development. We summarize current evidences that support the existence of a continuum between ageing and PD and justify the use of a Geroscience approach to study PD. We focus in particular on the role of inflammaging, the chronic, low-grade inflammation characteristic of elderly physiology, which can propagate and transmit both locally and systemically. We then describe PROPAG-AGEING design, which is based on the multi-omic characterization of peripheral samples from clinically characterized drug-naïve and advanced PD, PD discordant twins, healthy controls and "super-controls", i.e. centenarians, who never showed clinical signs of motor disability, and their offspring. Omic results are then validated in a large number of samples, including in vitro models of dopaminergic neurons and healthy siblings of PD patients, who are at higher risk of developing PD, with the final aim of identifying the molecular perturbations that can deviate the trajectories of healthy ageing towards PD development.
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http://dx.doi.org/10.1016/j.mad.2020.111426DOI Listing
March 2021

Plasma metabolome and cognitive skills in Down syndrome.

Sci Rep 2020 06 26;10(1):10491. Epub 2020 Jun 26.

Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Unit of Histology, Embryology and Applied Biology, University of Bologna, Via Belmeloro 8, 40126, Bologna, BO, Italy.

Trisomy 21 (Down syndrome, DS) is the main human genetic cause of intellectual disability (ID). Lejeune hypothesized that DS could be considered a metabolic disease, and we found that subjects with DS have a specific plasma and urinary metabolomic profile. In this work we confirmed the alteration of mitochondrial metabolism in DS and also investigated if metabolite levels are related to cognitive aspects of DS. We analyzed the metabolomic profiles of plasma samples from 129 subjects with DS and 46 healthy control (CTRL) subjects by H Nuclear Magnetic Resonance (NMR). Multivariate analysis of the NMR metabolomic profiles showed a clear discrimination (up to 94% accuracy) between the two groups. The univariate analysis revealed a significant alteration in 7 metabolites out of 28 assigned unambiguously. Correlations among the metabolite levels in DS and CTRL groups were separately investigated and statistically significant relationships appeared. On the contrary, statistically significant correlations among the NMR-detectable part of DS plasma metabolome and the different intelligence quotient ranges obtained by Griffiths-III or WPPSI-III tests were not found. Even if metabolic imbalance provides a clear discrimination between DS and CTRL groups, it appears that the investigated metabolomic profiles cannot be associated with the degree of ID.
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http://dx.doi.org/10.1038/s41598-020-67195-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319960PMC
June 2020

Diauxie and co-utilization of carbon sources can coexist during bacterial growth in nutritionally complex environments.

Nat Commun 2020 06 19;11(1):3135. Epub 2020 Jun 19.

Department of Biology, University of Florence, Florence, Italy.

It is commonly thought that when multiple carbon sources are available, bacteria metabolize them either sequentially (diauxic growth) or simultaneously (co-utilization). However, this view is mainly based on analyses in relatively simple laboratory settings. Here we show that a heterotrophic marine bacterium, Pseudoalteromonas haloplanktis, can use both strategies simultaneously when multiple possible nutrients are provided in the same growth experiment. The order of nutrient uptake is partially determined by the biomass yield that can be achieved when the same compounds are provided as single carbon sources. Using transcriptomics and time-resolved intracellular H-C NMR, we reveal specific pathways for utilization of various amino acids. Finally, theoretical modelling indicates that this metabolic phenotype, combining diauxie and co-utilization of substrates, is compatible with a tight regulation that allows the modulation of assimilatory pathways.
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http://dx.doi.org/10.1038/s41467-020-16872-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305145PMC
June 2020

Distal Unfolding of Ferricytochrome c Induced by the F82K Mutation.

Int J Mol Sci 2020 Mar 20;21(6). Epub 2020 Mar 20.

Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Florence, Italy.

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82-conserved amino acid and a key actor in cytochrome c intermolecular interactions-with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization.
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http://dx.doi.org/10.3390/ijms21062134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139943PMC
March 2020

Fingerprinting Alzheimer's Disease by H Nuclear Magnetic Resonance Spectroscopy of Cerebrospinal Fluid.

J Proteome Res 2020 04 12;19(4):1696-1705. Epub 2020 Mar 12.

Laboratory of Clinical Neurochemistry, Department of Medicine, Section of Neurology, University of Perugia, Perugia 06123, Italy.

In this study, we sought for a cerebrospinal fluid (CSF) metabolomic fingerprint in Alzheimer's disease (AD) patients characterized, according to the clinical picture and CSF AD core biomarkers (Aβ, p-tau, and t-tau), both at pre-dementia (mild cognitive impairment due to AD, MCI-AD) and dementia stages (ADdem) and in a group of patients with a normal CSF biomarker profile (non-AD) using untargeted H nuclear magnetic resonance (NMR) spectroscopy-based metabolomics. This is a retrospective study based on two independent cohorts: a Dutch cohort, which comprises 20 ADdem, 20 MCI-AD, and 20 non-AD patients, and an Italian cohort, constituted by 14 ADdem and 12 non-AD patients. H NMR CSF spectra were analyzed using OPLS-DA. Metabolomic fingerprinting in the Dutch cohort provides a significant discrimination (86.1% accuracy) between ADdem and non-AD. MCI-AD patients show a good discrimination with respect to ADdem (70.0% accuracy) but only slight differences when compared with non-AD (59.6% accuracy). Acetate, valine, and 3-hydroxyisovalerate result to be altered in ADdem patients. Valine correlates with cognitive decline at follow-up ( = 0.53, = 0.0011). The discrimination between ADdem and non-AD was confirmed in the Italian cohort. The CSF metabolomic fingerprinting shows a signature characteristic of ADdem patients with respect to MCI-AD and non-AD patients.
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http://dx.doi.org/10.1021/acs.jproteome.9b00850DOI Listing
April 2020

Iron Biomineral Growth from the Initial Nucleation Seed in L-Ferritin.

Chemistry 2020 May 31;26(26):5770-5773. Epub 2020 Mar 31.

Magnetic Resonance Center (CERM), University of Florence, Sesto Fiorentino, 50019, Italy.

X-ray structures of homopolymeric human L-ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (μ -oxo)tris[(μ -glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the level of the protein surface, capable of contributing coordination bonds and electrostatic guidance.
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http://dx.doi.org/10.1002/chem.202000064DOI Listing
May 2020

Effect of Estrogen Receptor Status on Circulatory Immune and Metabolomics Profiles of HER2-Positive Breast Cancer Patients Enrolled for Neoadjuvant Targeted Chemotherapy.

Cancers (Basel) 2020 Jan 29;12(2). Epub 2020 Jan 29.

Immunopathology and Cancer Biomarkers Unit, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, 33081 Aviano, Italy.

HER2-positive breast cancer (BC) represents a heterogeneous cancer disease. In an attempt to identify new stratification models useful for prognosis and therapeutic strategy, we investigated the influence of estrogen receptor (ER) status on the host immune and metabolomics profile of HER2-positive BC patients enrolled for neoadjuvant targeted chemotherapy (NATC). The study enrolled 43 HER2-positive BC patients eligible for NATC based on the trastuzumab-paclitaxel combination. Baseline circulatory cytokines and H NMR plasma metabolomics profiles were investigated. Differences in the immune cytokines and metabolomics profile as a function of the ER status, and their association with clinical outcomes were studied by multivariate and univariate analysis. Baseline metabolomics profiles were found to discriminate HER2-positive ER(+) from ER(-) BC patients. Within the ER(+) group an immune-metabolomics model, based on TNF-α and valine, predicted pathological complete response to NATC with 90.9% accuracy (AUROC = 0.92, = 0.004). Moreover, metabolomics information integrated with IL-2 and IL-10 cytokine levels were prognostic of relapse with an accuracy of 95.5%. The results indicate that in HER2-positive BC patients the ER status influences the host circulatory immune-metabolomics profile. The baseline immune-metabolomics assessment in combination with ER status could represent an independent stratification tool able to predict NATC response and disease relapse of HER2-positive patients.
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http://dx.doi.org/10.3390/cancers12020314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072610PMC
January 2020

H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma.

Cell Commun Signal 2019 08 28;17(1):108. Epub 2019 Aug 28.

IRCCS Neuromed, Pozzilli, IS, Italy.

Background: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway.

Methods: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice.

Results: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition.

Conclusions: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.
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http://dx.doi.org/10.1186/s12964-019-0421-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712882PMC
August 2019

Pre-analytical processes in medical diagnostics: New regulatory requirements and standards.

N Biotechnol 2019 Sep 15;52:121-125. Epub 2019 May 15.

Diagnostic- and Research Center for Molecular Biomedicine, Inst. of Pathology, Medical University of Graz, Neue Stiftingtalstr. 6, 8010 Graz, Austria. Electronic address:

In May 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. It will also affect health institutions developing and using in-house devices. The IVDR also has implications with respect to product performance validation and verification including the pre-analytics of biological samples used by IVD developers and diagnostic service providers. In parallel to the IVDR, a series of standards on pre-analytical sample processing has been published by the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN). These standards describe pre-analytical requirements for various types of analyses in various types of biospecimens. They are of relevance for IVD product developers in the context of (re)certification under the IVDR and to some extent also to devices manufactured and used only within health institutions. This review highlights the background and the rational for the pre-analytical standards. It describes the procedure that leads to these standards, the major implications of the standards and the requirements on pre-analytical workflows. In addition, it discusses the relationship between the standards and the IVDR.
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http://dx.doi.org/10.1016/j.nbt.2019.05.002DOI Listing
September 2019

Effect of the point mutation H54N on the ferroxidase process of Rana catesbeiana H' ferritin.

J Inorg Biochem 2019 08 7;197:110697. Epub 2019 May 7.

Department of Biotechnology, Chemistry and Pharmacy, Department of Excellence 2018-2020, University of Siena, via Aldo Moro, 2, 53110 Siena, Italy; Magnetic Resonance Center CERM, University of Florence, via Luigi Sacconi, 6, 50019 Sesto Fiorentino, FI, Italy. Electronic address:

Human H and Rana catesbeiana H' subunits in vertebrate ferritin protein cages catalyze the Fe(II) oxidation by molecular oxygen and promote the ferric oxide biomineral synthesis. By depositing iron biomineral, ferritins also prevent potentially toxic reactions products from Fe(II)-based Fenton chemistry. Recent work from our laboratory was aimed to describe the iron pathways within ferritin, from entrance into the cage to the ferroxidase site, and to understand the role played by amino-acid residues in iron trafficking and catalysis. Our approach exploits anomalous X-ray diffraction from ferritin crystals, exposed to a ferrous salt, to track transient iron binding sites along the path towards a well-defined di-iron site where they get oxidized by oxygen. Coupling structure determination with solution kinetic measurements on selected variants, allows validating the role played by key residues on the catalytic iron oxidation. Our previous studies on H' ferritin indicated the regulatory role played by His54, and by its human counterpart Gln58, on guiding Fe(II) ions to the catalytic site. Here, we have investigated the effects induced by substituting the wild type His54 with Asn54, having different iron coordination properties. We have obtained a series of atomic-resolution crystal structures that provide time-dependent snapshots of iron bound at different locations in the H' ferritin H54N variant. The comparison with H' ferritin and H' ferritin H54Q variant leads to identify a new iron binding site. Our kinetic and structural data support the role of H' ferritin residue 54 in regulating the access of Fe(II) ions to the catalytic site.
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http://dx.doi.org/10.1016/j.jinorgbio.2019.110697DOI Listing
August 2019

NMR for sample quality assessment in metabolomics.

N Biotechnol 2019 Sep 22;52:25-34. Epub 2019 Apr 22.

Center of Magnetic Resonance (CERM), University of Florence, Sesto Fiorentino FI, Italy; Department of Chemistry, University of Florence, Sesto Fiorentino FI, Italy. Electronic address:

The EU Framework 7 project SPIDIA was the occasion for development of NMR approaches to evaluate the impact of different pre-analytical treatments on the quality of biological samples dedicated to metabolomics. Systematic simulation of different pre-analytical procedures was performed on urine and blood serum and plasma. Here we review the key aspects of these studies that have led to the development of CEN technical specifications, to be translated into ISO/IS in the course of the EU Horizon 2020 project SPIDIA4P. Inspired by the SPIDIA results, follow-up research was performed, extending the analysis to different sample types and to the different effects of long-term storage. The latter activity was in conjunction with the local European da Vinci Biobank. These results (which partially contributed to the ANNEX of CEN/TS 16945"MOLECULAR IN VITRO DIAGNOSTIC EXAMINATIONS - SPECIFICATIONS FOR PRE-EXAMINATION PROCESSES FOR METABOLOMICS IN URINE, VENOUS BLOOD SERUM AND PLASMA") are presented in detail.
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http://dx.doi.org/10.1016/j.nbt.2019.04.004DOI Listing
September 2019

About the use of C-C NOESY in bioinorganic chemistry.

J Inorg Biochem 2019 03 8;192:25-32. Epub 2018 Dec 8.

CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy; Department of Chemistry "Ugo Schiff", University of Florence Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. Electronic address:

Herein we present examples of the application of the C-C Nuclear Overhauser Effect Spectroscopy (NOESY) experiment to the study of metalloproteins and we critically discuss the advantages and drawbacks of the method as a function of the molecular size of the investigated systems. The contribution is focused on a few case studies among the systems analyzed in the group of the corresponding author. The C-C NOESY experiment represents the gold standard for the observation of NMR signals in the 480 kDa ferritin nanocage and for monitoring its interaction with iron. By decreasing the protein size, the experiment progressively loses its importance as a tool for the detection of the complete spin pattern of the amino acid side chains, as exemplified by nickel-dependent regulatory protein, NikR (molecular mass of the homo-tetramer ~80 kDa). In very small proteins, such as mitochondrial cytochrome c (12.3 kDa), we are only able to detect cross peaks between adjacent C nuclei; this feature turned out to be useful for the assignment of the C core resonances of the porphyrin in a uniformly enriched heme.
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http://dx.doi.org/10.1016/j.jinorgbio.2018.12.006DOI Listing
March 2019

Creation and Characterization of a Genomically Hybrid Strain in the Nitrogen-Fixing Symbiotic Bacterium Sinorhizobium meliloti.

ACS Synth Biol 2018 10 3;7(10):2365-2378. Epub 2018 Oct 3.

Department of Biology , University of Florence , 50019 Sesto Fiorentino , Italy.

Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbor a multipartite genome composed of more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti. We term this strain as cis-hybrid, since it is produced by genomic material coming from the same species' pangenome. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S. meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extracellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modeling were employed for in silico prediction of metabolic flux reorganization. Phenotypes of the cis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with the cis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.
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http://dx.doi.org/10.1021/acssynbio.8b00158DOI Listing
October 2018

High-Throughput Metabolomics by 1D NMR.

Angew Chem Int Ed Engl 2019 01 11;58(4):968-994. Epub 2018 Nov 11.

CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.

Metabolomics deals with the whole ensemble of metabolites (the metabolome). As one of the -omic sciences, it relates to biology, physiology, pathology and medicine; but metabolites are chemical entities, small organic molecules or inorganic ions. Therefore, their proper identification and quantitation in complex biological matrices requires a solid chemical ground. With respect to for example, DNA, metabolites are much more prone to oxidation or enzymatic degradation: we can reconstruct large parts of a mammoth's genome from a small specimen, but we are unable to do the same with its metabolome, which was probably largely degraded a few hours after the animal's death. Thus, we need standard operating procedures, good chemical skills in sample preparation for storage and subsequent analysis, accurate analytical procedures, a broad knowledge of chemometrics and advanced statistical tools, and a good knowledge of at least one of the two metabolomic techniques, MS or NMR. All these skills are traditionally cultivated by chemists. Here we focus on metabolomics from the chemical standpoint and restrict ourselves to NMR. From the analytical point of view, NMR has pros and cons but does provide a peculiar holistic perspective that may speak for its future adoption as a population-wide health screening technique.
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http://dx.doi.org/10.1002/anie.201804736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391965PMC
January 2019

Cancer cell death induced by ferritins and the peculiar role of their labile iron pool.

Oncotarget 2018 Jun 15;9(46):27974-27984. Epub 2018 Jun 15.

Center for Magnetic Resonance, University of Florence, Florence, Italy.

Cellular uptake of human H-ferritin loaded with 50 or 350 iron ions results in significant cytotoxicity on HeLa cells at submicromolar concentrations. Conversely, Horse Spleen Ferritin, that can be considered a model of L-cages, as it contains only about 10% of H subunits, even when loaded with 1000 iron ions, is toxic only at >1 order of magnitude higher protein concentrations. We propose here that the different cytotoxicity of the two ferritin cages originates from the presence in H-ferritin of a pool of non-biomineralized iron ions bound at the ferroxidase catalytic sites of H-ferritin subunits. This iron pool is readily released during the endosomal-mediated H-ferritin internalization.
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http://dx.doi.org/10.18632/oncotarget.25416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021343PMC
June 2018

Plasma and urinary metabolomic profiles of Down syndrome correlate with alteration of mitochondrial metabolism.

Sci Rep 2018 02 14;8(1):2977. Epub 2018 Feb 14.

Neonatology Unit, St. Orsola-Malpighi Polyclinic, Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Via Massarenti 9, 40138, Bologna, BO, Italy.

Down syndrome (DS) is caused by the presence of a supernumerary copy of the human chromosome 21 (Hsa21) and is the most frequent genetic cause of intellectual disability (ID). Key traits of DS are the distinctive facies and cognitive impairment. We conducted for the first time an analysis of the Nuclear Magnetic Resonance (NMR)-detectable part of the metabolome in plasma and urine samples, studying 67 subjects with DS and 29 normal subjects as controls selected among DS siblings. Multivariate analysis of the NMR metabolomic profiles showed a clear discrimination (up to of 80% accuracy) between the DS and the control groups. The univariate analysis of plasma and urine revealed a significant alteration for some interesting metabolites. Remarkably, most of the altered concentrations were consistent with the 3:2 gene dosage model, suggesting effects caused by the presence of three copies of Hsa21 rather than two: DS/normal ratio in plasma was 1.23 (pyruvate), 1.47 (succinate), 1.39 (fumarate), 1.33 (lactate), 1.4 (formate). Several significantly altered metabolites are produced at the beginning or during the Krebs cycle. Accounting for sex, age and fasting state did not significantly affect the main result of both multivariate and univariate analysis.
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http://dx.doi.org/10.1038/s41598-018-20834-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813015PMC
February 2018

Investigation of the Iron(II) Release Mechanism of Human H-Ferritin as a Function of pH.

J Chem Inf Model 2017 09 12;57(9):2112-2118. Epub 2017 Sep 12.

Magnetic Resonance Center (CERM), University of Florence , Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy.

We investigated the kinetics of the release of iron(II) ions from the internal cavity of human H-ferritin as a function of pH. Extensive molecular dynamics simulations of the entire 24-mer ferritin provided atomic-level information on the release mechanism. Double protonation of His residues at pH 4 facilitates the removal of the iron ligands within the C3 channel through the formation of salt bridges, resulting in a significantly lower release energy barrier than pH 9.
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http://dx.doi.org/10.1021/acs.jcim.7b00306DOI Listing
September 2017

Unsaturated Long-Chain Fatty Acids Are Preferred Ferritin Ligands That Enhance Iron Biomineralization.

Chemistry 2017 Jul 27;23(41):9879-9887. Epub 2017 Jun 27.

Istituto per lo Studio delle Macromolecole, CNR, Via Corti 12, 20133, Milano, Italy.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.
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http://dx.doi.org/10.1002/chem.201701164DOI Listing
July 2017

Structural basis of mitochondrial dysfunction in response to cytochrome phosphorylation at tyrosine 48.

Proc Natl Acad Sci U S A 2017 04 27;114(15):E3041-E3050. Epub 2017 Mar 27.

Instituto de Investigaciones Químicas, cicCartuja, Universidad de Sevilla-Spanish National Scientific Council (CSIC), 41092 Seville, Spain;

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome phosphorylation-in particular, at tyrosine 48-is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome by replacing tyrosine 48 with -carboxy-methyl-l-phenylalanine (CMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around CMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome and to develop novel therapeutic approaches based on its prosurvival effects.
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http://dx.doi.org/10.1073/pnas.1618008114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393209PMC
April 2017

NMR metabolomics highlights sphingosine kinase-1 as a new molecular switch in the orchestration of aberrant metabolic phenotype in cancer cells.

Mol Oncol 2017 05 30;11(5):517-533. Epub 2017 Mar 30.

CERM and Department of Chemistry, University of Florence, Italy.

Strong experimental evidence in animal and cellular models supports a pivotal role of sphingosine kinase-1 (SK1) in oncogenesis. In many human cancers, SK1 levels are upregulated and these increases are linked to poor prognosis in patients. Here, by employing untargeted NMR-based metabolomic profiling combined with functional validations, we report the crucial role of SK1 in the metabolic shift known as the Warburg effect in A2780 ovarian cancer cells. Indeed, expression of SK1 induced a high glycolytic rate, characterized by increased levels of lactate along with increased expression of the proton/monocarboxylate symporter MCT1, and decreased oxidative metabolism, associated with the accumulation of intermediates of the tricarboxylic acid cycle and reduction in CO production. Additionally, SK1-expressing cells displayed a significant increase in glucose uptake paralleled by GLUT3 transporter upregulation. The role of SK1 is not limited to the induction of aerobic glycolysis, affecting metabolic pathways that appear to support the biosynthesis of macromolecules. These findings highlight the role of SK1 signaling axis in cancer metabolic reprogramming, pointing out innovative strategies for cancer therapies.
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http://dx.doi.org/10.1002/1878-0261.12048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527469PMC
May 2017

Chemistry at the protein-mineral interface in L-ferritin assists the assembly of a functional (μ-oxo)Tris[(μ-peroxo)] triiron(III) cluster.

Proc Natl Acad Sci U S A 2017 03 15;114(10):2580-2585. Epub 2017 Feb 15.

Centro di Risonanze Magnetiche, University of Florence, Sesto Fiorentino, Florence 50019, Italy;

X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (μ-oxo)Tris[(μ-peroxo)(μ-glutamato-κ:κ')](glutamato-κ)(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin.
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http://dx.doi.org/10.1073/pnas.1614302114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347543PMC
March 2017

Insights into Interprotein Electron Transfer of Human Cytochrome Variants Arranged in Multilayer Architectures by Means of an Artificial Silica Nanoparticle Matrix.

ACS Omega 2016 Dec 1;1(6):1058-1066. Epub 2016 Dec 1.

Institute of Applied Life Sciences, Biosystems Technology, Technical University of Applied Sciences Wildau, Hochschulring 1, 15745 Wildau, Germany.

The redox behavior of proteins plays a crucial part in the design of bioelectronic systems. We have demonstrated several functional systems exploiting the electron exchange properties of the redox protein cytochrome (cyt ) in combination with enzymes and photoactive proteins. The operation is based on an effective reaction at modified electrodes but also to a large extent on the capability of self-exchange between cyt molecules in a surface-fixed state. In this context, different variants of human cyt have been examined here with respect to an altered heterogeneous electron transfer (ET) rate in a monolayer on electrodes as well as an enhanced self-exchange rate while being incorporated in multilayer architectures. For this purpose, mutants of the wild-type (WT) protein have been prepared to change the chemical nature of the surface contact area near the heme edge. The structural integrity of the variants has been verified by NMR and UV-vis measurements. It is shown that the single-point mutations can significantly influence the heterogeneous ET rate at thiol-modified gold electrodes and that electroactive protein/silica nanoparticle multilayers can be constructed with all forms of human cyt prepared. The kinetic behavior of electron exchange for the mutant proteins in comparison with that of the WT has been found altered in some multilayer arrangements. Higher self-exchange rates have been found for K79A. The results demonstrate that the position of the introduced change in the charge situation of cyt has a profound influence on the exchange behavior. In addition, the behavior of the cyt variants in assembled multilayers is found to be rather similar to the situation of cyt self-exchange in solution verified by NMR.
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http://dx.doi.org/10.1021/acsomega.6b00213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044710PMC
December 2016

Ferroxidase Activity in Eukaryotic Ferritin is Controlled by Accessory-Iron-Binding Sites in the Catalytic Cavity.

Chemistry 2016 Nov 21;22(45):16213-16219. Epub 2016 Sep 21.

CERM and Department of Chemistry, University of Florence, Via Sacconi 6 Sesto Fiorentino, Firenze, 50019, Italy.

Ferritins are iron-storage nanocage proteins that catalyze the oxidation of Fe to Fe at ferroxidase sites. By a combination of structural and spectroscopic techniques, Asp140, together with previously identified Glu57 and Glu136, is demonstrated to be an essential residue to promote the iron oxidation at the ferroxidase site. However, the presence of these three carboxylate moieties in close proximity to the catalytic centers is not essential to achieve binding of the Fe substrate to the diferric ferroxidase sites with the same coordination geometries as in the wild-type cages.
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http://dx.doi.org/10.1002/chem.201602842DOI Listing
November 2016

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels.

J Biol Chem 2016 Dec 18;291(49):25617-25628. Epub 2016 Oct 18.

From the Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126 Pisa,

Ferritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe-loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H' protein and some of its variants as case studies. We describe the structural features that determine Fe translocation with atomistic detail, and we propose a putative mechanism for Fe transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin.
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http://dx.doi.org/10.1074/jbc.M116.748046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207259PMC
December 2016

Individual Human Metabolic Phenotype Analyzed by (1)H NMR of Saliva Samples.

J Proteome Res 2016 06 3;15(6):1787-93. Epub 2016 May 3.

Medical University Graz , Auenbruggerplatz 2, A-8036 Graz, Austria.

Saliva is an important physiological fluid that contains a complex mixture of analytes that may produce a characteristic individual signature. In recent years, it has been demonstrated that urine possesses a clear signature of the individual metabolic phenotype. Here NMR-based metabolomics was employed to analyze saliva from 23 healthy volunteers. About six saliva samples were collected daily from each individual for 10 consecutive days: 7 days in a real-life situation (days 1-7, Phase I) and 3 days (days 8-10, Phase II) under a standardized diet plus a physical exercise program at day 10. The result is the first demonstration of the existence of an individual metabolic phenotype in saliva. A systematic comparative analysis with urine samples from the same collection scheme demonstrates that the individual phenotype in saliva is slightly weaker than that in urine but less influenced by diet.
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http://dx.doi.org/10.1021/acs.jproteome.5b01060DOI Listing
June 2016

Solid-State NMR of PEGylated Proteins.

Angew Chem Int Ed Engl 2016 Feb 12;55(7):2446-9. Epub 2016 Jan 12.

Magnetic Resonance Center (CERM) and Department of Chemistry "Ugo Schiff", University of Florence, and Magnetic Resonance Consortium (CIRMMP), Via L. Sacconi 6, 50019, Sesto Fiorentino, FI, Italy.

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.
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http://dx.doi.org/10.1002/anie.201510148DOI Listing
February 2016

Multi-omic profiles of human non-alcoholic fatty liver disease tissue highlight heterogenic phenotypes.

Sci Data 2015 Dec 8;2:150068. Epub 2015 Dec 8.

Medical Faculty, Institute for Stem Cell Research and Regenerative Medicine, Heinrich Heine University, 40225 Düsseldorf, Germany.

Non-alcoholic fatty liver disease (NAFLD) is a consequence of sedentary life style and high fat diets with an estimated prevalence of about 30% in western countries. It is associated with insulin resistance, obesity, glucose intolerance and drug toxicity. Additionally, polymorphisms within, e.g., APOC3, PNPLA3, NCAN, TM6SF2 and PPP1R3B, correlate with NAFLD. Several studies have already investigated later stages of the disease. This study explores the early steatosis stage of NAFLD with the aim of identifying molecular mechanisms underlying the etiology of NAFLD. We analyzed liver biopsies and serum samples from patients with high- and low-grade steatosis (also pre-disease states) employing transcriptomics, ELISA-based serum protein analyses and metabolomics. Here, we provide a detailed description of the various related datasets produced in the course of this study. These datasets may help other researchers find new clues for the etiology of NAFLD and the mechanisms underlying its progression to more severe disease states.
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http://dx.doi.org/10.1038/sdata.2015.68DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672680PMC
December 2015