Publications by authors named "Diego Sbardella"

44 Publications

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

Proteasome inhibition by bortezomib parallels a reduction in head and neck cancer cells growth, and an increase in tumor-infiltrating immune cells.

Sci Rep 2021 Sep 24;11(1):19051. Epub 2021 Sep 24.

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

Head and neck cancer (HNC) has frequently an aggressive course for the development of resistance to standard chemotherapy. Thus, the use of innovative therapeutic drugs is being assessed. Bortezomib is a proteasome inhibitor with anticancer effects. In vitro antitumoral activity of Bortezomib was investigated employing human tongue (SCC-15, CAL-27), pharynx (FaDu), salivary gland (A-253) cancer cell lines and a murine cell line (SALTO-5) originated from a salivary gland adenocarcinoma arising in BALB-neuT male mice transgenic for the oncogene neu. Bortezomib inhibited cell proliferation, triggered apoptosis, modulated the expression and activation of pro-survival signaling transduction pathways proteins activated by ErbB receptors and inhibited proteasome activity in vitro. Intraperitoneal administration of Bortezomib delayed tumor growth of SALTO-5 cells transplanted in BALB-neuT mice, protracted mice survival and adjusted tumor microenvironment by increasing tumor-infiltrating immune cells (CD4 and CD8 T cells, B lymphocytes, macrophages, and Natural Killer cells) and by decreasing vessels density. In addition, Bortezomib modified the expression of proteasome structural subunits in transplanted SALTO-5 cells. Our findings further support the use of Bortezomib for the treatment of HNC and reveal its ineffectiveness in counteracting the activation of deregulated specific signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed.
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http://dx.doi.org/10.1038/s41598-021-98450-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8463577PMC
September 2021

Dexamethasone Downregulates Autophagy through Accelerated Turn-Over of the Ulk-1 Complex in a Trabecular Meshwork Cells Strain: Insights on Steroid-Induced Glaucoma Pathogenesis.

Int J Mol Sci 2021 May 31;22(11). Epub 2021 May 31.

IRCCS-Fondazione Bietti, 00198 Rome, Italy.

Steroid-induced glaucoma is a severe pathological condition, sustained by a rapidly progressive increase in intraocular pressure (IOP), which is diagnosed in a subset of subjects who adhere to a glucocorticoid (GC)-based therapy. Molecular and clinical studies suggest that either natural or synthetic GCs induce a severe metabolic dysregulation of Trabecular Meshwork Cells (TMCs), an endothelial-derived histotype with phagocytic and secretive functions which lay at the iridocorneal angle in the anterior segment of the eye. Since TMCs physiologically regulate the composition and architecture of trabecular meshwork (TM), which is the main outflow pathway of aqueous humor, a fluid which shapes the eye globe and nourishes the lining cell types, GCs are supposed to trigger a pathological remodeling of the TM, inducing an IOP increase and retina mechanical compression. The metabolic dysregulation of TMCs induced by GCs exposure has never been characterized at the molecular detail. Herein, we report that, upon dexamethasone exposure, a TMCs strain develops a marked inhibition of the autophagosome biogenesis pathway through an enhanced turnover of two members of the Ulk-1 complex, the main platform for autophagy induction, through the Ubiquitin Proteasome System (UPS).
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http://dx.doi.org/10.3390/ijms22115891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198647PMC
May 2021

Citicoline in Ophthalmological Neurodegenerative Disease: A Comprehensive Review.

Pharmaceuticals (Basel) 2021 Mar 20;14(3). Epub 2021 Mar 20.

IRCCS-Fondazione Bietti, Via Livenza, 3, 00198 Rome, Italy.

Cytidine 5'-diphosphocholine has been widely studied in systemic neurodegenerative diseases, like Alzheimer's disease, Parkinson's disease, and brain ischemia. The rationale for the use of citicoline in ophthalmological neurodegenerative diseases, including glaucoma, anterior ischemic optic neuropathy, and diabetic retinopathy, is founded on its multifactorial mechanism of action and the involvement in several metabolic pathways, including phospholipid homeostasis, mitochondrial dynamics, as well as cholinergic and dopaminergic transmission, all being involved in the complexity of the visual transmission. This narrative review is aimed at reporting both pre-clinical data regarding the involvement of citicoline in such metabolic pathways (including new insights about its role in the intracellular proteostasis through an interaction with the proteasome) and its effects on clinical psychophysical, electrophysiological, and morphological outcomes following its use in ophthalmological neurodegenerative diseases (including the results of the most recent prospective randomized clinical trials).
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http://dx.doi.org/10.3390/ph14030281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003774PMC
March 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

Glaucoma and neuroinflammation: An overview.

Surv Ophthalmol 2021 Sep-Oct;66(5):693-713. Epub 2021 Feb 11.

IRCCS - Fondazione Bietti, Rome, Italy.

Glaucoma is an optic neuropathy characterized by well-defined optic disc morphological changes (i.e., cup enlargement, neuroretinal border thinning, and notching, papillary vessel modifications) consequent to retinal ganglion cell loss, axonal degeneration, and lamina cribrosa remodeling. These modifications tend to be progressive and are the main cause of functional damage in glaucoma. Despite the latest findings about the pathophysiology of the disease, the exact trigger mechanisms and the mechanism of degeneration of retinal ganglion cells and their axons have not been completely elucidated. Neuroinflammation may play a role in both the development and the progression of the disease as a result of its effects on retinal environment and retinal ganglion cells. We summarize the latest findings about neuroinflammation in glaucoma and examine the connection between risk factors, neuroinflammation, and retinal ganglion cell degeneration.
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http://dx.doi.org/10.1016/j.survophthal.2021.02.003DOI Listing
February 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

Role of proteolytic enzymes in the COVID-19 infection and promising therapeutic approaches.

Biochem Pharmacol 2020 12 19;182:114225. Epub 2020 Sep 19.

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

In the Fall of 2019 a sudden and dramatic outbreak of a pulmonary disease (Coronavirus Disease COVID-19), due to a new Coronavirus strain (i.e., SARS-CoV-2), emerged in the continental Chinese area of Wuhan and quickly diffused throughout the world, causing up to now several hundreds of thousand deaths. As for common viral infections, the crucial event for the viral life cycle is the entry of genetic material inside the host cell, realized by the spike protein of the virus through its binding to host receptors and its activation by host proteases; this is followed by translation of the viral RNA into a polyprotein, exploiting the host cell machinery. The production of individual mature viral proteins is pivotal for replication and release of new virions. Several proteolytic enzymes either of the host and of the virus act in a concerted fashion to regulate and coordinate specific steps of the viral replication and assembly, such as (i) the entry of the virus, (ii) the maturation of the polyprotein and (iii) the assembly of the secreted virions for further diffusion. Therefore, proteases involved in these three steps are important targets, envisaging that molecules which interfere with their activity are promising therapeutic compounds. In this review, we will survey what is known up to now on the role of specific proteolytic enzymes in these three steps and of most promising compounds designed to impair this vicious cycle.
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http://dx.doi.org/10.1016/j.bcp.2020.114225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501082PMC
December 2020

Structural and functional evidence for citicoline binding and modulation of 20S proteasome activity: Novel insights into its pro-proteostatic effect.

Biochem Pharmacol 2020 07 13;177:113977. Epub 2020 Apr 13.

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

Citicoline or CDP-choline is a drug, made up by a cytidine 5'-diphosphate moiety and choline, which upon adsorption is rapidly hydrolyzed into cytidine 5'-diphosphate and choline, easily bypassing the blood-brain barrier. Once in the brain, these metabolites are used to re-synthesize citicoline in neurons and in the other cell histo-types which uptake them. Citicoline administration finds broad therapeutic application in the treatment of glaucoma as well as other retinal disorders by virtue of its safety profile and neuro-protective and neuroenhancer activity, which significantly improves the visual function. Further, though supported by limited clinical studies, this molecule finds therapeutic application in neurodegenerative disease, delaying the cognitive decline in Alzheimer's Disease (AD) and Parkinson's Disease (PD) subjects. In this work we show that citicoline greatly affects the proteolytic activity of the 20S proteasome on synthetic and natural substrates, functioning as a bimodal allosteric modulator, likely binding at multiple sites. In silico binding simulations identify several potential binding sites for citicoline on 20S proteasome, and their topology envisages the possibility that, by occupying some of these pockets, citicoline may induce a conformational shift of the 20S proteasome, allowing to sketch a working hypothesis for the structural basis of its function as allosteric modulator. In addition, we show that over the same concentration range citicoline affects the distribution of assembled proteasome populations and turn-over of ubiquitinated proteins in SH-SY5Y and SK-N-BE human neuroblastoma cells, suggesting its potential role as a regulator of proteostasis in nervous cells.
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http://dx.doi.org/10.1016/j.bcp.2020.113977DOI Listing
July 2020

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

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

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

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

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

Effects of oral administration of common antioxidant supplements on the energy metabolism of red blood cells. Attenuation of oxidative stress-induced changes in Rett syndrome erythrocytes by CoQ10.

Mol Cell Biochem 2020 Jan 8;463(1-2):101-113. Epub 2019 Oct 8.

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

Nutritional supplements are traditionally employed for overall health and for managing some health conditions, although controversies are found concerning the role of antioxidants-mediated benefits in vivo. Consistently with its critical role in systemic redox buffering, red blood cell (RBC) is recognized as a biologically relevant target to investigate the effects of oxidative stress. In RBC, reduction of the ATP levels and adenylate energy charge brings to disturbance in intracellular redox status. In the present work, several popular antioxidant supplements were orally administrated to healthy adults and examined for their ability to induce changes on the energy metabolism and oxidative status in RBC. Fifteen volunteers (3 per group) were treated for 30 days per os with epigallocatechin gallate (EGCG) (1 g green tea extract containing 50% EGCG), resveratrol (325 mg), coenzyme Q10 (CoQ10) (300 mg), vitamin C (1 g), and vitamin E (400 U.I.). Changes in the cellular levels of high-energy compounds (i.e., ATP and its catabolites, NAD and GTP), GSH, GSSG, and malondialdehyde (MDA) were simultaneously analyzed by ion-pairing HPLC. Response to oxidative stress was further investigated through the oxygen radical absorptive capacity (ORAC) assay. According to our experimental approach, (i) CoQ10 appeared to be the most effective antioxidant inducing a high increase in ATP/ADP, ATP/AMP, GSH/GSSG ratio and ORAC value and, in turn, a reduction of NAD concentration, (ii) EGCG modestly modulated the intracellular energy charge potential, while (iii) Vitamin E, vitamin C, and resveratrol exhibited very weak effects. Given that, the antioxidant potential of CoQ10 was additionally assessed in a pilot study which considered individuals suffering from Rett syndrome (RTT), a severe X-linked neuro-developmental disorder in which RBC oxidative damages provide biological markers for redox imbalance and chronic hypoxemia. RTT patients (n = 11), with the typical clinical form, were supplemented for 12 months with CoQ10 (300 mg, once daily). Level of lipid peroxidation (MDA production) and energy state of RBCs were analyzed at 2 and 12 months. Our data suggest that CoQ10 may significantly attenuate the oxidative stress-induced damage in RTT erythrocytes.
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http://dx.doi.org/10.1007/s11010-019-03633-5DOI Listing
January 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

Reductive nitrosylation of ferric microperoxidase-11.

J Biol Inorg Chem 2019 02 2;24(1):21-29. Epub 2018 Nov 2.

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

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c, which is considered as a heme-protein model. Here, the reductive nitrosylation of ferric MP11 (MP11(III)) under anaerobic conditions has been investigated between pH 7.4 and 9.2, at T = 20.0 °C. At pH ≤ 7.7, NO binds reversibly to MP11(III) leading to the formation of the MP11(III)-NO complex. However, between pH 8.2 and 9.2, the addition of NO to MP11(III) leads to the formation of ferrous nitrosylated MP11(II) (MP11(II)-NO). In fact, the transient MP11{FeNO} species is converted to ferrous deoxygenated MP11 (MP11(II)) by OH- and HO-based catalysis, which represents the rate-limiting step of the whole reaction. Then, MP11(II) binds NO very rapidly leading to MP11(II)-NO formation. Over the whole pH range explored, the apparent values of k, k, and K (= k/k) for MP11(III)(-NO) (de)nitrosylation are essentially pH independent, ranging between 5.8 × 10 M s and 1.6 × 10 M s, between 1.9 s and 3.7 s, and between 1.4 × 10 M and 4.6 × 10 M, respectively. Values of the apparent pseudo-first-order rate constant for the MP11{FeNO} conversion to MP11(II) (i.e., h) increase linearly with pH; the apparent values [Formula: see text] and [Formula: see text] are 7.2 × 10 M s and 2.5 × 10 s, respectively. Present data confirm that MP11 is a useful molecular model to highlight the role of the protein matrix on the heme-based reactivity.
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http://dx.doi.org/10.1007/s00775-018-1623-zDOI Listing
February 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 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

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site.

PLoS One 2018 15;13(2):e0192651. Epub 2018 Feb 15.

CNR Institute for Molecular Recognition, Roma Italy.

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813964PMC
April 2018

Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages.

ChemMedChem 2018 03 14;13(5):422-430. Epub 2018 Feb 14.

European Research Centre for Drug Discovery and Development - NatSynDrugs - and Department of Biotechnology, Chemistry, and Pharmacy, University of Siena, via Aldo Moro 1, 53100, Siena, Italy.

The enzyme Zmp1 is a zinc-containing peptidase that plays a critical role in the pathogenicity of Mycobacterium tuberculosis. Herein we describe the identification of a small set of Zmp1 inhibitors based on a novel 8-hydroxyquinoline-2-hydroxamate scaffold. Among the synthesized compounds, N-(benzyloxy)-8-hydroxyquinoline-2-carboxamide (1 c) was found to be the most potent Zmp1 inhibitor known to date, and its binding mode was analyzed both by kinetics studies and molecular modeling, identifying critical interactions of 1 c with the zinc ion and residues in the active site. The effect of 1 c on intracellular Mycobacterium survival was assayed in J774 murine macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and human monocyte-derived macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and genotoxicity were also assessed. Overall, inhibitor 1 c displays interesting in vitro antitubercular properties worthy of further investigation.
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http://dx.doi.org/10.1002/cmdc.201700759DOI Listing
March 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

Retention of Mitochondria in Mature Human Red Blood Cells as the Result of Autophagy Impairment in Rett Syndrome.

Sci Rep 2017 09 26;7(1):12297. Epub 2017 Sep 26.

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

Rett Syndrome (RTT), which affects approximately 1:10.000 live births, is a X-linked pervasive neuro-developmental disorder which is caused, in the vast majority of cases, by a sporadic mutation in the Methyl-CpG-binding protein-2 (MeCP2) gene. This is a transcriptional activator/repressor with presumed pleiotropic activities. The broad tissue expression of MeCP2 suggests that it may be involved in several metabolic pathways, but the molecular mechanisms which provoke the onset and progression of the syndrome are largely unknown. In this paper, we report that primary fibroblasts that have been isolated from RTT patients display a defective formation of autophagosomes under conditions of nutrient starvation and that the mature Red Blood Cells of some RTT patients retain mitochondria. Moreover, we provide evidence regarding the accumulation of the p62/SQSTM1 protein and ubiquitin-aggregated structures in the cerebellum of Mecp2 knockout mouse model (Mecp2 ) during transition from the non-symptomatic to the symptomatic stage of the disease. Hence, we propose that a defective autophagy could be involved in the RTT clinical phenotype, which introduces new molecular perspectives in the pathogenesis of the syndrome.
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http://dx.doi.org/10.1038/s41598-017-12069-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614985PMC
September 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

Oxygen exchange and energy metabolism in erythrocytes of Rett syndrome and their relationships with respiratory alterations.

Mol Cell Biochem 2017 Feb 7;426(1-2):205-213. Epub 2017 Jan 7.

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

Rett syndrome (RTT) is a neurodevelopmental disorder, mainly affecting females, which is associated to a mutation on the methyl-CpG-binding protein 2 gene. In the pathogenesis and progression of classic RTT, red blood cell (RBC) morphology has been shown to be an important biosensor for redox imbalance and chronic hypoxemia. Here we have evaluated the impact of oxidation and redox imbalance on several functional properties of RTT erythrocytes. In particular, we report for the first time a stopped-flow measurement of the kinetics of oxygen release by RBCs and the analysis of the intrinsic affinity of the hemoglobin (Hb). According to our experimental approach, RBCs from RTT patients do not show any intrinsic difference with respect to those from healthy controls neither in Hb's oxygen-binding affinity nor in O exchange processes at 37 °C. Therefore, these factors do not contribute to the observed alteration of the respiratory function in RTT patients. Moreover, the energy metabolism of RBCs, from both RTT patients and controls, was evaluated by ion-pairing HPLC method and related to the level of malondialdehyde and to the oxidative radical scavenging capacity of red cells. Results have clearly confirmed significant alterations in antioxidant defense capability, adding important informations concerning the high-energy compound levels in RBCs of RTT subjects, underlying possible correlations with inflammatory tissue alterations.
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http://dx.doi.org/10.1007/s11010-016-2893-9DOI Listing
February 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

Cyanide binding to ferrous and ferric microperoxidase-11.

J Biol Inorg Chem 2016 07 26;21(4):511-22. Epub 2016 May 26.

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

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cytc). MP11 is characterized by a covalently linked solvent-exposed heme group, the heme-Fe atom being axially coordinated by a histidyl residue. Here, the reactions of ferrous and ferric MP11 (MP11-Fe(II) and MP11-Fe(III), respectively) with cyanide have been investigated from the kinetic and thermodynamic viewpoints, at pH 7.0 and 20.0 °C. Values of the second-order rate constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 4.5 M(-1) s(-1) and 8.9 × 10(3) M(-1) s(-1), respectively. Values of the first-order rate constant for cyanide dissociation from ligated MP11-Fe(II) and MP11-Fe(III) are 1.8 × 10(-1) s(-1) and 1.5 × 10(-3) s(-1), respectively. Values of the dissociation equilibrium constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 3.7 × 10(-2) and 1.7 × 10(-7) M, respectively, matching very well with those calculated from kinetic parameters so that no intermediate species seem to be involved in the ligand-binding process. The pH-dependence of cyanide binding to MP11-Fe(III) indicates that CN(-) is the only binding species. Present results have been analyzed in parallel with those of several heme-proteins, suggesting that (1) the ligand accessibility to the metal center and cyanide ionization may modulate the formation of heme-Fe-cyanide complexes, and (2) the general polarity of the heme pocket and/or hydrogen bonding of the heme-bound ligand may affect cyanide exit from the protein matrix. Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c. Penta-coordinated MP11 displays a very high reactivity towards cyanide, whereas the reactivity of hexa-coordinated horse heart cytochrome c is very low.
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http://dx.doi.org/10.1007/s00775-016-1361-zDOI Listing
July 2016

The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin.

J Biol Inorg Chem 2016 06 24;21(3):421-32. Epub 2016 Mar 24.

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

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4], whereas the value of k on for NO2 (-) reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M(-1) s(-1) (at pH 7.4). CL facilitates the NO2 (-)-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 (-)-mediated conversion of CL-CM-cytc-Fe(II) to CL-CM-cytc-Fe(II)-NO (5.6 ± 0.6 M(-1) s(-1); at pH 7.4) being slightly higher than that for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO (2.6 ± 0.3 M(-1) s(-1); at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10(-6) M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are -1.05 ± 0.07 and -1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH(-). These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL-CM-cytc.
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http://dx.doi.org/10.1007/s00775-016-1351-1DOI Listing
June 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

NO2(-)-mediated nitrosylation of ferrous microperoxidase-11.

J Inorg Biochem 2015 Dec 4;153:121-127. Epub 2015 Jul 4.

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

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cyt c) and characterized by a covalently-linked solvent-exposed heme group. Here, kinetics of the NO2(-)-mediated nitrosylation of ferrous MP11 (MP11-Fe(II)) is reported. Data were obtained between pH6.4 and 8.2, at 20.0°C. The NO2(-)-mediated conversion of MP11-Fe(II) to MP11-Fe(II)-NO requires one proton; accordingly, values of the apparent second-order rate constant (kon) decrease by about two orders of magnitude from (2.9±0.3)×10(1)M(-1)s(-1) to (5.0±0.6)×10(-1)M(-1)s(-1) upon increasing pH from 6.4 to pH8.2. The slope of the linear fitting of Logkon versus pH is -1.00±0.06. Values of kon for the NO2(-)-mediated nitrosylation of MP11-Fe(II) are similar to those of penta-coordinated cardiolipin-bound horse heart cyt c, exceeding by about two orders of magnitude those of wild-type horse heart cyt c. Present results highlight the role of heme distal residues in modulating horse heart cyt c reactivity.
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http://dx.doi.org/10.1016/j.jinorgbio.2015.06.022DOI Listing
December 2015

Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

PLoS One 2015 17;10(7):e0132455. Epub 2015 Jul 17.

Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Via Montpellier 1, I-00133, Rome, Italy; Center for Space Biomedicine, University of Roma Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy.

Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132455PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506093PMC
May 2016

α-dystroglycan is a potential target of matrix metalloproteinase MMP-2.

Matrix Biol 2015 Jan 4;41:2-7. Epub 2014 Dec 4.

Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy. Electronic address:

Dystroglycan (DG) is a member of the glycoprotein complex associated to dystrophin and composed by two subunits, the β-DG, a transmembrane protein, and the α-DG, an extensively glycosylated extracellular protein. The β-DG ectodomain degradation by the matrix metallo-proteinases (i.e., MMP-2 and MMP-9) in both, pathological and physiological conditions, has been characterized in detail in previous publications. Since the amounts of α-DG and β-DG at the cell surface decrease when gelatinases are up-regulated, we investigated the degradation of α-DG subunit by MMP-2. Present data show, for the first time, that the proteolysis of α-DG indeed occurs on a native glycosylated molecule enriched from rabbit skeletal muscle. In order to characterize the α-DG portion, which is more prone to cleavage by MMP-2, we performed different degradations on tailored recombinant domains of α-DG spanning the whole subunit. The overall bulk of results casts light on a relevant susceptibility of the α-DG to MMP-2 degradation with particular reference to its C-terminal domain, thus opening a new scenario on the role of gelatinases (in particular of MMP-2) in the degradation of this glycoprotein complex, taking place in the course of pathological processes.
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http://dx.doi.org/10.1016/j.matbio.2014.11.007DOI Listing
January 2015
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