Publications by authors named "Grazia R Tundo"

26 Publications

  • Page 1 of 1

At the Cutting Edge against Cancer: A Perspective on Immunoproteasome and Immune Checkpoints Modulation as a Potential Therapeutic Intervention.

Cancers (Basel) 2021 Sep 28;13(19). Epub 2021 Sep 28.

Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, 00133 Rome, Italy.

Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers.
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http://dx.doi.org/10.3390/cancers13194852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507813PMC
September 2021

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

Cooperative Binding of the Cationic Porphyrin Tris-T4 Enhances Catalytic Activity of 20S Proteasome Unveiling a Complex Distribution of Functional States.

Int J Mol Sci 2020 Sep 29;21(19). Epub 2020 Sep 29.

Dipartimento di Farmacia, Università di Napoli "Federico II", Via D. Montesano 49, 80131 Napoli, Italy.

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri -methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.
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http://dx.doi.org/10.3390/ijms21197190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582714PMC
September 2020

Ferric nitrosylated myoglobin catalyzes peroxynitrite scavenging.

J Biol Inorg Chem 2020 05 14;25(3):361-370. Epub 2020 Mar 14.

Dipartimento odi Scienze Cliniche e Medicina Translazionale, Università di Roma "Tor Vergata", Via Montpellier 1, 00133, Rome, Italy.

Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., k) is (6.8 ± 0.7) × 10 M s and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., k) is 3.1 ± 0.3 s. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., k/k = (4.6 ± 0.7) × 10 M) is virtually the same as that directly measured (i.e., K = (3.8 ± 0.5) × 10 M). In the absence of NO, Mb(III) catalyzes the conversion of peroxynitrite to NO, the value of the second-order rate constant (i.e., k) being (1.9 ± 0.2) × 10 M s. However, in the presence of NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (k* = (2.8 ± 0.3) × 10 M s). These data expand the multiple roles of NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO.
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http://dx.doi.org/10.1007/s00775-020-01767-2DOI Listing
May 2020

Kinetics of cyanide and carbon monoxide dissociation from ferrous human haptoglobin:hemoglobin(II) complexes.

J Biol Inorg Chem 2020 05 7;25(3):351-360. Epub 2020 Mar 7.

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

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) trapping the αβ dimers of Hb. In turn, the Hp:Hb complexes display heme-based reactivity. Here, the kinetics of cyanide and carbon monoxide dissociation from ferrous-ligated Hp:Hb complexes are reported at pH 7.0 and 20.0 °C. Cyanide dissociation from Hp1-1:Hb(II)-CN and Hp2-2:Hb-CN has been followed upon the dithionite-mediated conversion of ferric to ferrous-ligated Hp:Hb complexes. Values of k for the dithionite-mediated reduction of Hp1-1:Hb(III)-CN and Hp2-2:Hb(III)-CN are (7.3 ± 1.1) × 10 M s and (6.2 ± 1.0) × 10 M s, respectively. Values of the first-order rate constant (i.e., h) for cyanide dissociation from Hp1-1:Hb(II)-CN and Hp2-2:Hb(II)-CN are (1.2 ± 0.2) × 10 s and (1.3 ± 0.2) × 10 s, respectively. CO dissociation from Hp:Hb(II)-CO complexes has been followed by replacing CO with NO. Values of the first-order rate constant (i.e., l) for CO dissociation from Hp1-1:Hb(II)-CO are (1.4 ± 0.2) × 10 s and (6.2 ± 0.8) × 10 s, and those from Hp2-2:Hb(II)-CO are (1.3 ± 0.2) × 10 s and (7.3 ± 0.9) × 10 s. Values of k, h, and l correspond to those reported for the R-state of tetrameric Hb and isolated α and β chains. This highlights the view that the conformation of the Hb αβ-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. Furthermore, unlike ferric Hb(III), ligated ferrous Hb(II) does not show an assembly-linked structural change.
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http://dx.doi.org/10.1007/s00775-020-01766-3DOI Listing
May 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

On the Horizon: Targeting Next-Generation Immune Checkpoints for Cancer Treatment.

Chemotherapy 2019 6;64(2):62-80. Epub 2019 Aug 6.

Laboratory of Molecular Oncology, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Rome, Italy.

Background: Immune checkpoints are critical regulatory pathways of the immune system which finely tune the response to biological threats. Among them, the CD-28/CTLA-4 and PD-1/PD-L1 axes play a key role in tumour immune escape and are well-established targets of cancer immunotherapy.

Summary: The clinical experience accumulated to date provides unequivocal evidence that anti-CTLA-4, PD-1, or PD-L1 monoclonal antibodies, used as monotherapy or in combination regimes, are effective in a variety of advanced/metastatic types of cancer, with improved clinical outcomes compared to conventional chemotherapy. However, the therapeutic success is currently restricted to a limited subset of patients and reliable predictive biomarkers are still lacking. Key Message: The identification and characterization of additional co-inhibitory pathways as novel pharmacological targets to improve the clinical response in refractory patients has led to the development of different immune checkpoint inhibitors, the activities of which are currently under investigation. In this review, we discuss recent literature data concerning the mechanisms of action of next-generation monoclonal antibodies targeting LAG-3, TIM-3, and TIGIT co-inhibitory molecules that are being explored in clinical trials, as single agents or in combination with other immune-stimulating agents.
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http://dx.doi.org/10.1159/000500902DOI Listing
November 2019

Insights into Proteasome Conformation Dynamics and Intersubunit Communication.

Trends Biochem Sci 2018 11 22;43(11):852-853. Epub 2018 Aug 22.

Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy. Electronic address:

A recently published paper applies cryo-electron microscopy (EM) studies and biochemical/genetic approaches for the elucidation of the mechanisms linking nucleotide binding by ATPases, proteasome conformation dynamics, and gate opening of the 20S core particle. These insights potentially represent a milestone in our understanding of the structural dynamics of the 26S proteasome.
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http://dx.doi.org/10.1016/j.tibs.2018.08.002DOI Listing
November 2018

The nitrite reductase activity of ferrous human hemoglobin:haptoglobin 1-1 and 2-2 complexes.

J Inorg Biochem 2018 10 20;187:116-122. Epub 2018 Jul 20.

Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Via Montpellier 1, I-00133 Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Via Celso Ulpiani 27, I-70126 Bari, Italy.

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http://dx.doi.org/10.1016/j.jinorgbio.2018.07.005DOI Listing
October 2018

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

Electrostatic Map Of Proteasome α-Rings Encodes The Design of Allosteric Porphyrin-Based Inhibitors Able To Affect 20S Conformation By Cooperative Binding.

Sci Rep 2017 12 6;7(1):17098. Epub 2017 Dec 6.

Dipartimento di Scienze Cliniche e Medicina Traslazionale, Università di Roma Tor Vergata, Via Montpellier 1, 00133, Roma, Italy.

The importance of allosteric proteasome inhibition in the treatment of cancer is becoming increasingly evident. Motivated by this urgent therapeutic need, we have recently identified cationic porphyrins as a highly versatile class of molecules able to regulate proteasome activity by interfering with gating mechanisms. In the present study, the mapping of electrostatic contacts bridging the regulatory particles with the α-rings of the human 20S proteasome led us to the identification of (meso-tetrakis(4-N-methylphenyl pyridyl)-porphyrin (pTMPyPP4) as a novel non-competitive inhibitor of human 20S proteasome. pTMPyPP4 inhibition mechanism implies a positive cooperative binding to proteasome, which disappears when a permanently open proteasome mutant (α-3ΔN) is used, supporting the hypothesis that the events associated with allosteric proteasome inhibition by pTMPyPP4 interfere with 20S gating and affect its "open-closed" equilibrium. Therefore, we propose that the spatial distribution of the negatively charged residues responsible for the interaction with regulatory particles at the α-ring surface of human 20S may be exploited as a blueprint for the design of allosteric proteasome regulators.
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http://dx.doi.org/10.1038/s41598-017-17008-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719074PMC
December 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

Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

FEBS J 2016 10 29;283(20):3755-3770. Epub 2016 Sep 29.

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

Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ) and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium.
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http://dx.doi.org/10.1111/febs.13841DOI Listing
October 2016

Cationic porphyrins are tunable gatekeepers of the 20S proteasome.

Chem Sci 2016 Feb 9;7(2):1286-1297. Epub 2015 Nov 9.

Dipartimento di Scienze Chimiche , Università di Catania , Viale Andrea Doria 6 , 95125 Catania , Italy . Email:

The 20S proteasome is a barrel-shaped enzymatic assembly playing a critical role in proteome maintenance. Access of proteasome substrates to the catalytic chamber is finely regulated through gating mechanisms which involve aromatic and negatively charged residues located at the N-terminal tails of α subunits. However, despite the importance of gates in regulating proteasome function, up to now very few molecules have been shown to interfere with the equilibrium by which the catalytic channel exchanges between the open and closed states. In this light, and inspired by previous results evidencing the antiproteasome potential of cationic porphyrins, here we combine experimental (enzyme kinetics, UV stopped flow and NMR) and computational (bioinformatic analysis and docking studies) approaches to inspect proteasome inhibition by -tetrakis(4--methylpyridyl)-porphyrin (HT4) and its two - and -isomers. We show that in a first, fast binding event HT4 accommodates in a pocket made of negatively charged and aromatic residues present in α1 (Asp10, Phe9), α3 (Tyr5), α5 (Asp9, Tyr8), α6 (Asp7, Tyr6) and α7 (Asp9, Tyr8) subunits thereby stabilizing the closed conformation. A second, slower binding mode involves interaction with the grooves which separate the α- from the β-rings. Of note, the proteasome inhibition by - and -HT4 decreases significantly if compared to the parent compound, thus underscoring the role played by spatial distribution of the four peripheral positive charges in regulating proteasome-ligand interactions. We think that our results may pave the way to further studies aimed at rationalizing the molecular basis of novel, and more sophisticated, proteasome regulatory mechanisms.
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http://dx.doi.org/10.1039/c5sc03312hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975898PMC
February 2016

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins.

PLoS One 2015 20;10(5):e0125005. Epub 2015 May 20.

Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy.

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2- binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2- concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2-binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125005PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439042PMC
February 2016

Novel Platinum(II) compounds modulate insulin-degrading enzyme activity and induce cell death in neuroblastoma cells.

J Biol Inorg Chem 2015 Jan 2;20(1):101-8. Epub 2014 Dec 2.

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

The properties of three novel Platinum(II) compounds toward the insulin-degrading enzyme (IDE) enzymatic activity have been investigated under physiological conditions. The rationale of this study resides on previous observations that these compounds, specifically designed and synthesized by some of us, induce apoptosis in various cancer cell lines, whereas IDE has been proposed as a putative oncogene involved in neuroblastoma onset and progression. Two of these compounds, namely [PtCl(O,O'-acac)(DMSO)] and [Pt(O,O'-acac)(γ-acac)(DMS)], display a modulatory behavior, wherefore activation or inhibition of IDE activity occurs over different concentration ranges (suggesting the existence of two binding sites on the enzyme). On the other hand, [Pt(O,O'-acac)(γ-acac)(DMSO)] shows a typical competitive inhibitory pattern, characterized by a meaningful affinity constant (K i  = 0.95 ± 0.21 μM). Although all three compounds induce cell death in neuroblastoma SHSY5Y cells at concentrations exceeding 2 μM, the two modulators facilitate cells' proliferation at concentrations ≤ 1.5 μM, whereas the competitive inhibitor [Pt(O,O'-acac)(γ-acac)(DMSO)] only shows a pro-apoptotic activity at all investigated concentrations. These features render the [Pt(O,O'-acac)(γ-acac)(DMSO)] a promising "lead compound" for the synthesis of IDE-specific inhibitors (not characterized yet) with therapeutic potentiality.
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http://dx.doi.org/10.1007/s00775-014-1217-3DOI Listing
January 2015

Role of metalloproteinases in tendon pathophysiology.

Mini Rev Med Chem 2014 ;14(12):978-87

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

Tendons play a crucial role in musculoskeletal functioning because they physically connect bones and muscles making the movement of articular joints possible. The molecular composition of tendons mostly include collagen I fibrils, which aggregate together to form fibers to form a fascicle. A complex network composed of resident cells (i.e., tenocytes) and extracellular matrix macromolecules (glycosaminoglycans, proteoglycans, glycoproteins and other non collagenous proteins) interact and define the structure of tendons and their properties. Development, renewal and remodeling of tendons composition occur at all ages of living organisms so the homeostasis of proteolytic systems is a critical issue. A major role is played by Metalloproteinases, a family of Zn(2+)-dependent endopeptidases involved in the catabolism of several components of the extracellular matrix, such as collagens, proteoglycans, fibronectin and many others. Among these, two main classes are mostly involved in tendon pathophysiology, namely the Matrix Metalloproteinases (MMPs) and a Disintegrin-like and Metalloproteinase domain with Thrombospondin motifs (ADAMTSs). This study analyses the various aspects of the roles played by Metalloproteinases in the physiological and pathological processes of tendons.
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http://dx.doi.org/10.2174/1389557514666141106132411DOI Listing
July 2015

Nitrosylation mechanisms of Mycobacterium tuberculosis and Campylobacter jejuni truncated hemoglobins N, O, and P.

PLoS One 2014 22;9(7):e102811. Epub 2014 Jul 22.

Department of Clinical Sciences and Translational Medicine, University of Roma "Tor Vergata", Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy.

Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tuberculosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102811PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106858PMC
December 2015

Warfarin modulates the nitrite reductase activity of ferrous human serum heme-albumin.

J Biol Inorg Chem 2013 Dec 15;18(8):939-46. Epub 2013 Sep 15.

Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, 00146, Rome, Italy,

Human serum heme-albumin (HSA-heme-Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA-heme-Fe [HSA-heme-Fe(II)] is reported. The value of the second-order rate constant for the reduction of [Formula: see text] to NO and the concomitant formation of nitrosylated HSA-heme-Fe(II) (i.e., k on) is 1.3 M(-1) s(-1) at pH 7.4 and 20 °C. Values of k on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA-heme-Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA-heme-Fe(II) is (3.1 ± 0.4) × 10(-4) M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the [Formula: see text] reductase activity of HSA-heme-Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(-heme-Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.
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http://dx.doi.org/10.1007/s00775-013-1040-2DOI Listing
December 2013

Reciprocal allosteric modulation of carbon monoxide and warfarin binding to ferrous human serum heme-albumin.

PLoS One 2013 21;8(3):e58842. Epub 2013 Mar 21.

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

Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058842PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605432PMC
October 2013

Functional and structural roles of the N-terminal extension in Methanosarcina acetivorans protoglobin.

Biochim Biophys Acta 2013 Sep 26;1834(9):1813-23. Epub 2013 Feb 26.

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

Functional and structural properties of protoglobin from Methanosarcina acetivorans, whose Cys(101)E20 residue was mutated to Ser (MaPgb*), and of mutants missing either the first 20 N-terminal amino acids (MaPgb*-ΔN20 mutant), or the first 33 N-terminal amino acids [N-terminal loop of 20 amino acids and a 13-residue Z-helix, preceding the globin fold A-helix; (MaPgb*-ΔN20Z mutant)] have been investigated. In keeping with the MaPgb*-ΔN20 mutant crystal structure, here reported at 2.0Å resolution, which shows an increased exposure of the haem propionates to the solvent, the analysis of ligand binding kinetics highlights high accessibility of ligands to the haem pocket in ferric MaPgb*-ΔN20. CO binding to ferrous MaPgb*-ΔN20 displays a marked biphasic behavior, with a fast binding process close to that observed in MaPgb* and a slow carbonylation process, characterized by a rate-limiting step. Conversely, removal of the first 33 residues induces a substantial perturbation of the overall MaPgb* structure, with loss of α-helical content and potential partial collapse of the protein chain. As such, ligand binding kinetics are characterized by very slow rates that are independent of ligand concentration, this being indicative of a high energy barrier for ligand access to the haem, possibly due to localized misfolding. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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http://dx.doi.org/10.1016/j.bbapap.2013.02.026DOI Listing
September 2013

Metal ions affect insulin-degrading enzyme activity.

J Inorg Biochem 2012 Dec 23;117:351-8. Epub 2012 Jun 23.

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

Insulin degradation is a finely tuned process that plays a major role in controlling insulin action and most evidence supports IDE (insulin-degrading enzyme) as the primary degradative agent. However, the biomolecular mechanisms involved in the interaction between IDE and its substrates are often obscure, rendering the specific enzyme activity quite difficult to target. On the other hand, biometals, such as copper, aluminum and zinc, have an important role in pathological conditions such as Alzheimer's disease or diabetes mellitus. The metabolic disorders connected with the latter lead to some metallostasis alterations in the human body and many studies point at a high level of interdependence between diabetes and several cations. We have previously reported (Grasso et al., Chem. Eur. J. 17 (2011) 2752-2762) that IDE activity toward Aβ peptides can be modulated by metal ions. Here, we have investigated the effects of different metal ions on the IDE proteolytic activity toward insulin as well as a designed peptide comprising a portion of the insulin B chain (B20-30), which has a very low affinity for metal ions. The results obtained by different experimental techniques clearly show that IDE is irreversibly inhibited by copper(I) but is still able to process its substrates when it is bound to copper(II).
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http://dx.doi.org/10.1016/j.jinorgbio.2012.06.010DOI Listing
December 2012

Evidence for pH-dependent multiple conformers in iron(II) heme-human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding.

J Biol Inorg Chem 2012 Jan 6;17(1):133-47. Epub 2011 Sep 6.

Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata", Via Montpellier 1, 00133 Rome, Italy.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.
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http://dx.doi.org/10.1007/s00775-011-0837-0DOI Listing
January 2012

Ibuprofen and warfarin modulate allosterically ferrous human serum heme-albumin nitrosylation.

Biochem Biophys Res Commun 2011 Jul 24;411(1):185-9. Epub 2011 Jun 24.

Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, I-00146 Roma, Italy.

Ferrous human serum heme-albumin (HSA-heme-Fe(II)) displays globin-like properties. Here, the effect of ibuprofen and warfarin on kinetics of HSA-heme-Fe(II) nitrosylation is reported. Values of the second-order rate constant for HSA-heme-Fe(II) nitrosylation (k(on)) decrease from 6.3 × 10(6)M(-1)s(-1) in the absence of drugs, to 4.1 × 10(5)M(-1)s(-1) and 4.8 × 10(5)M(-1)s(-1), in the presence of saturating amounts of ibuprofen and warfarin, respectively, at pH 7.0 and 20.0°C. From the dependence of k(on) on the drug concentration, values of the dissociation equilibrium constant for ibuprofen and warfarin binding to HSA-heme-Fe(II) (i.e., K=3.2 × 10(-3)M and 2.6 × 10(-4)M, respectively) were determined. The observed allosteric effects could indeed reflect ibuprofen and warfarin binding to the regulatory fatty acid binding site FA2, which brings about an alteration of heme coordination, slowing down HSA-heme-Fe(II) nitrosylation. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.
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http://dx.doi.org/10.1016/j.bbrc.2011.06.130DOI Listing
July 2011

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme-albumin.

J Biol Inorg Chem 2011 Jan 25;16(1):97-108. Epub 2010 Sep 25.

Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Viale Guglielmo Marconi 446, Rome, Italy.

Human serum heme-albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA-heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K (0) = (3.9 ± 0.4) × 10(-4) and (1.3 ± 0.1) × 10(-5) M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K (h) = (4.3 ± 0.4) × 10(-3) and (1.2 ± 0.1) × 10(-4) M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H (0) = (1.9 ± 0.2) × 10(-8) M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H (d) = (2.1 ± 0.2) × 10(-7) M]. In the absence and presence of CO(2), the values of the second-order rate constant (l (on)) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10(5) and 4.3 × 10(5) M(-1) s(-1), respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO(2). Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free L: -tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow's site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow's site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.
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http://dx.doi.org/10.1007/s00775-010-0706-2DOI Listing
January 2011

Somatostatin: a novel substrate and a modulator of insulin-degrading enzyme activity.

J Mol Biol 2009 Feb 25;385(5):1556-67. Epub 2008 Nov 25.

Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Rome, Italy.

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes beta-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward beta-amyloid. In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k(cat) (s(-1))=0.38 (+/-0.05); K(m) (M)=7.5 (+/-0.9) x 10(-6)] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic beta-amyloid through a decrease of the K(m) toward this substrate, which corresponds to the 10-25 amino acid sequence of the Abeta(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn(2+) ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic beta-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.
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http://dx.doi.org/10.1016/j.jmb.2008.11.025DOI Listing
February 2009
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