CNR Institute of Protein Biochemistry


CNR Institute of Protein Biochemistry

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Capasso C.,CNR Institute of Protein Biochemistry | Supuran C.T.,University of Florence
Journal of Enzyme Inhibition and Medicinal Chemistry | Year: 2014

Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families. © 2014 Informa UK Ltd.

Pan P.,University of Tampere | Vermelho A.B.,Federal University of Rio de Janeiro | Capaci Rodrigues G.,Federal University of Rio de Janeiro | Scozzafava A.,University of Florence | And 4 more authors.
Journal of Medicinal Chemistry | Year: 2013

An α-carbonic anhydrase (CA, EC has been identified, cloned, and characterized from the unicellular protozoan Trypanosoma cruzi, the causative agent of Chagas disease. The enzyme (TcCA) has a very high catalytic activity for the CO2 hydration reaction, being similar kinetically to the human (h) isoform hCA II, although it is devoid of the His64 proton shuttle. A large number of aromatic/heterocyclic sulfonamides and some 5-mercapto-1,3,4-thiadiazoles were investigated as TcCA inhibitors. The aromatic sulfonamides were weak inhibitors (KI values of 192 nM to 84 μM), whereas some heterocyclic compounds inhibited the enzyme with KI values in the range 61.6-93.6 nM. The thiols were the most potent in vitro inhibitors (KI values of 21.1-79.0 nM), and some of them also inhibited the epimastigotes growth of two T. cruzi strains in vivo. © 2013 American Chemical Society.

Del Prete S.,CNR Institute of Protein Biochemistry | Vullo D.,University of Florence | Scozzafava A.,University of Florence | Capasso C.,CNR Institute of Protein Biochemistry | Supuran C.T.,University of Florence
Bioorganic and Medicinal Chemistry | Year: 2014

We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO2 hydration, its physiological reaction, with a kcat of 1.3 × 105 s-1 and a kcat/KM of 3.3 × 107 M-1 s-1. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (KIs >200 mM) whereas other halides and pseudohalides were submillimolar-millimolar inhibitors (KIs in the range of 0.93-8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KIs in the range of 9-90 μM, whereas acetazolamide inhibited the enzyme with a KI of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO2 fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO2 capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms. © 2013 Published by Elsevier Ltd. All rights reserved.

Di Fiore A.,CNR Institute of Biostructure and Bioimaging | Capasso C.,CNR Institute of Protein Biochemistry | De Luca V.,CNR Institute of Protein Biochemistry | Monti S.M.,CNR Institute of Biostructure and Bioimaging | And 6 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

SspCA, a novel 'extremo-α-carbonic anhydrase' isolated from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1, is an efficient catalyst for the hydration of CO2 and presents exceptional thermostability. Indeed, SspCA retains a high catalytic activity even after being heated to 343-373 14;K for several hours. Here, the crystallographic structure of this α-carbonic anhydrase (α-CA) is reported and the factors responsible for its function at high temperature are elucidated. In particular, the study suggests that increased structural compactness, together with an increased number of charged residues on the protein surface and a greater number of ionic networks, seem to be the key factors involved in the higher thermostability of this enzyme with respect to its mesophilic homologues. These findings are of extreme importance, since they provide a structural basis for the understanding of the mechanisms responsible for thermal stability in the α-CA family for the first time. The data obtained offer a tool that can be exploited to engineer α-CAs in order to obtain enzymes with enhanced thermostability for use in the harsh conditions of the CO2 capture and sequestration processes. © 2013 International Union of Crystallography.

Cocca E.,CNR Institute of Protein Biochemistry | Iorio S.D.,The Second University of Naples | Capriglione T.,University of Naples Federico II
Molecular Phylogenetics and Evolution | Year: 2011

Rolling-circle (RC) eukaryotic transposons, known as helitrons, are found in a wide range of organisms, from protist to mammals. Autonomous helitrons have a distinctive open reading frame (ORF) encoding a polypeptide that contains typical domains for RC replication (RCR): the Rep (RCR initiator) and the DNA helicase domains. These elements are believed to have an important role in the host genome evolution, owing to their frequent capture of host genes, some of which can evolve into novel genes or become essential for helitron transposition. We conducted a molecular analysis of the suborder Notothenioidei, a group of Perciformes that currently dominate the Antarctic waters by virtue of their remarkable cold-adaptation ability. A novel helitron from the genome of the icefish species Chionodraco hamatus, belonging to the Channichthyidae, the most derived Notothenioids family, was isolated, characterized and designated as HeliNoto (8.9. kb). Its ORF was compared to homologous sequences from different species in a comprehensive phylogenetic analysis. For the first time the putative functional domains of a helitron were subjected to a well accurate structural analysis including chromosomal localization. Finally, the distribution of HeliNoto among Notothenioids was investigated. © 2011 Elsevier Inc.

Castellano I.,CNR Institute of Protein Biochemistry | Merlino A.,University of Naples Federico II | Rossi M.,CNR Institute of Protein Biochemistry | La Cara F.,CNR Institute of Protein Biochemistry
Biochimie | Year: 2010

Gamma-glutamyltranspeptidases (γ-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, γ-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce γ-glutamyl compounds. To find out γ-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. γ-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using l-glutamic acid γ-(4-nitroanilide) as the substrate reveals that the active enzyme has K m 7.6 μM and V max 0.36 μmol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 °C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other γ-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized γ-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability. © 2010 Elsevier Masson SAS.

De Matteis M.A.,Telethon Institute of Genetics and Medicine | Wilson C.,Telethon Institute of Genetics and Medicine | D'Angelo G.,CNR Institute of Protein Biochemistry
BioEssays | Year: 2013

Initially identified as a key phosphoinositide that controls membrane trafficking at the Golgi complex, phosphatidylinositol-4-phosphate (PI4P) has emerged as a key molecule in the regulation of a diverse array of cellular functions. In this review we will discuss selected examples of the findings that in the last few years have significantly increased our awareness of the regulation and roles of PI4P in the Golgi complex and beyond. We will also highlight the role of PI4P in infection and cancer. We believe that, with the increasing number of regulators and effectors of PI4P identified, the time is ripe for a more integrated approach of study. A first step in this direction is the delineation of PI4P-centered molecular networks that we provide using data from low and high throughput studies in yeast and mammals. © 2013 WILEY Periodicals, Inc.

Raj D.A.A.,CNR Institute of Protein Biochemistry | Raj D.A.A.,The Second University of Naples | Fiume I.,CNR Institute of Protein Biochemistry | Capasso G.,The Second University of Naples | Pocsfalvi G.,CNR Institute of Protein Biochemistry
Kidney International | Year: 2012

Urinary exosomes have received considerable attention as a potential biomarker source for the diagnosis of renal diseases. Notwithstanding, their use in protein biomarker research is hampered by the lack of efficient methods for vesicle isolation, lysis, and protein quantification. Here we report an improved ultracentrifugation-based method that facilitates the solubilization and removal of major impurities associated with urinary exosomes. A double-cushion sucrose/D 2 O centrifugation step was used after a two-step differential centrifugation to separate exosomes from the heavier vesicles. After the removal of uromodulin, 378 and 79 unique proteins were identified, respectively, in low-and high-density fractions. Comparison of our data with two previously published data sets helped to define proteins commonly found in urinary exosomes. Lysis, protein extraction, and in-solution digestion of exosomes were then optimized for MudPIT application. More than a hundred exosomal proteins were quantified by four-plex iTRAQ analysis of single and pooled samples from two different age groups. For healthy men, six proteins (TSN1, PODXL, IDHC, PPAP, ACBP, and ANXA5) showed significant expression differences between exosome pools of those aged 25-50 and 50-70 years old. Thus, exosomes isolated by our method provide the basis for the development of robust quantitative methods for protein biomarker research. © 2012 International Society of Nephrology.

Boraschi D.,CNR Institute of Protein Biochemistry | Italiani P.,CNR Institute of Neuroscience
Immunology Letters | Year: 2014

The immune system of the elderly is particularly susceptible to infectious diseases and displays reduced response to vaccination. The current vaccines, designed for young and adult individuals, proved less effective and less protective in old people. The world population is rapidly ageing, and consequently preventing infectious diseases in the elderly have become an important public health issue. To this end, it is necessary to develop novel vaccines especially suited to raising protective immunity in the ageing population. Approaches in this direction include high-dose vaccines, booster vaccinations, different immunisation routes, and use of new adjuvants. These approaches, still empirical, must be supported by intensive research to unravel the biological and molecular mechanisms underlying immunosenescence. Only this knowledge would allow us to design approaches to immune rejuvenation and more effective vaccines for protecting the elderly. © 2014 Elsevier B.V.

Nazzaro F.,CNR Institute of Food Sciences | Orlando P.,CNR Institute of Protein Biochemistry | Fratianni F.,CNR Institute of Food Sciences | Coppola R.,CNR Institute of Food Sciences
Current Opinion in Biotechnology | Year: 2012

Microencapsulation can represent an excellent example of microtechnologies applied to food science and biotechnology. Microencapsulation can be successfully applied to entrap natural compounds, like essential oils or vegetal extracts containing polyphenols with well known antimicrobial properties to be used in food packaging. Microencapsulation preserves lactic acid bacteria, both starters and probiotics, in food and during the passage through the gastrointestinal tract, and may contribute to the development of new functional foods. © 2011 Elsevier Ltd.

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