Publications by authors named "Gabriella Angiulli"

5 Publications

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New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins.

Elife 2020 03 3;9. Epub 2020 Mar 3.

Laboratory of Molecular Electron Microscopy, Rockefeller University, New York, United States.

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.
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http://dx.doi.org/10.7554/eLife.53530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053995PMC
March 2020

Inhibition of Leishmania infantum trypanothione reductase by diaryl sulfide derivatives.

J Enzyme Inhib Med Chem 2017 Dec;32(1):304-310

b Istituto di Biologia e Patologia Molecolari - CNR , and Dipartimento di Scienze Biochimiche , " Sapienza" Università di Roma , Roma , Italia.

The study presented here aimed at identifying a new class of compounds acting against Leishmania parasites, the causative agent of Leishmaniasis. For this purpose, the thioether derivatives of our in-house library have been evaluated in whole-cell screening assays in order to determine their in vitro activity against Leishmania protozoan. Among them, promising results have been achieved with compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine) (IC=29.43 µM), which is able to impair the mechanism of the parasite defence against the reactive oxygen species by inhibiting the trypanothione reductase (TR) with high efficiency (K 0.25 ± 0.18 µM). The X-ray structure of L. infantum TR in complex with RDS 777 disclosed the mechanism of action of this compound that binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding and avoiding its reduction.
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http://dx.doi.org/10.1080/14756366.2016.1250755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010130PMC
December 2017

Leishmania infantum trypanothione reductase is a promiscuous enzyme carrying an NADPH:O2 oxidoreductase activity shared by glutathione reductase.

Biochim Biophys Acta 2015 Sep 29;1850(9):1891-7. Epub 2015 May 29.

Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, Rome Italy. Electronic address:

Background: Leishmania infantum is a protozoan of the trypanosomatid family causing visceral leishmaniasis. Leishmania parasites are transmitted by the bite of phlebotomine sand flies to the human host and are phagocyted by macrophages. The parasites synthesize N1-N8-bis(glutationyl)-spermidine (trypanothione, TS2), which furnishes electrons to the tryparedoxin-tryparedoxin peroxidase couple to reduce the reactive oxygen species produced by macrophages. Trypanothione is kept reduced by trypanothione reductase (TR), a FAD-containing enzyme essential for parasite survival.

Methods: The enzymatic activity has been studied by stopped-flow, absorption spectroscopy, and amperometric measurements.

Results: The study reported here demonstrates that the steady-state parameters change as a function of the order of substrates addition to the TR-containing solution. In particular, when the reaction is carried out by adding NADPH to a solution containing the enzyme and trypanothione, the KM for NADPH decreases six times compared to the value obtained by adding TS2 as last reagent to start the reaction (1.9 vs. 12μM). More importantly, we demonstrate that TR is able to catalyze the oxidation of NADPH also in the absence of trypanothione. Thus, TR catalyzes the reduction of O2 to water through the sequential formation of C(4a)-(hydro)peroxyflavin and sulfenic acid intermediates. This NADPH:O2 oxidoreductase activity is shared by Saccharomyces cerevisiae glutathione reductase (GR).

Conclusions: TR and GR, in the absence of their physiological substrates, may catalyze the electron transfer reaction from NADPH to molecular oxygen to yield water.

General Significance: TR and GR are promiscuous enzymes.
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http://dx.doi.org/10.1016/j.bbagen.2015.05.022DOI Listing
September 2015

Structure-based discovery of the first non-covalent inhibitors of Leishmania major tryparedoxin peroxidase by high throughput docking.

Sci Rep 2015 May 7;5:9705. Epub 2015 May 7.

1] European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, via Aldo Moro 2, 53100, Siena [2] Dip. di Biotecnologie, Chimica e Farmacia, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.

Leishmaniasis is a neglected vector-born disease caused by a protozoan of the genus Leishmania and affecting more than 1.300.000 people worldwide. The couple tryparedoxin/tryparedoxin peroxidase is essential for parasite survival in the host since it neutralizes the hydrogen peroxide produced by macrophages during the infection. Herein we report a study aimed at discovering the first class of compounds able to non-covalently inhibit tryparedoxin peroxidase. We have solved the high-resolution structure of Tryparedoxin peroxidase I from Leishmania major (LmTXNPx) in the reduced state and in fully folded conformation. A first series of compounds able to inhibit LmTXNPx was identified by means of the high throughput docking technique. The inhibitory activity of these compounds was validated by a Horseradish peroxidase-based enzymatic assay and their affinity for LmTXNPx calculated by surface plasmon resonance experiments. On the basis of these results, the analysis of the enzyme-inhibitor docked models allowed us to rationally design and synthesize a series of N,N-disubstituted 3-aminomethyl quinolones. These compounds showed an inhibitory potency against LmTXNPx in the micromolar range. Among them, compound 12 represents the first non-covalent LmTXNPx inhibitor reported to date and could pave the way to the discovery of a new class of drugs against leishmaniasis.
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http://dx.doi.org/10.1038/srep09705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423475PMC
May 2015

Targeting polyamine metabolism for finding new drugs against leishmaniasis: a review.

Mini Rev Med Chem 2015 ;15(3):243-52

CNR- Istituto di Biologia e Patologia Molecolari (IBPM), c/o Dipartimento di Scienze Biochimiche, Sapienza University of Roma, P.le A. Moro 5, 00185 Roma, Italy.

Leishmaniasis is a neglected disease affecting more than 12 million people worldwide. The most used drugs are pentavalent antimonials that are very toxic and display the problem of drug resistance, especially in endemic regions such as Bihar in India. For this reason, it is urgent to find new and less toxic drugs against leishmaniasis. To this end, the understanding of pathways affecting parasite survival is of prime importance for targeted drug discovery. The parasite survival inside the macrophage is strongly dependent on polyamine metabolism. Polyamines are, in fact, very important for cell growth and proliferation. In particular, spermidine (Spd), the final product of the polyamine biosynthesis pathway, serves as a precursor for trypanothione (N1,N8- bis(glutathionyl)spermidine, T(SH)2) and hypusine (N(ε)-(4-amino-2-hydroxybutyl)lysine). T(SH)2 is a key molecule for parasite defense against the hydrogen peroxide produced by macrophages during the infection. Hypusination is a posttranslational modification occurring exclusively in the eukaryotic initiation factor 5A (eIF5A), which has an important role in avoiding the ribosome stalling during the biosynthesis of protein containing polyprolines sequences. The enzymes, belonging to the spermidine metabolism, i.e. arginase (ARG), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase (SpdS), trypanothione synthetase (TryS or TSA), trypanothione reductase (TryR or TR), tryparedoxin peroxidase (TXNPx), deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are promising targets for the development of new drugs against leishmaniasis. This minireview furnishes a picture of the structural, functional and inhibition studies on polyamine metabolism enzymes that could guide the discovery of new drugs against leishmaniasis.
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http://dx.doi.org/10.2174/138955751503150312141044DOI Listing
December 2015