Paolo Bernardi

Università degli Studi di Padova, Dipartimento di Scienze Biomediche, Faculty Member

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Looking Back to the Future of Mitochondrial Research

Frontiers in Physiology

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Mitochondrial Permeability Transition

Cells

The mitochondrial permeability transition (PT) is a phenomenon that can be broadly defined as an ... more

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Mitochondrial H+ permeability through the ADP/ATP carrier

Nature Metabolism, 2019

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New Perspectives in Mitochondrial Research

… mitochondrial cyclosporin A-sensitive permeability transition pore. I. Evidence for two separate Me2+ binding sites with opposing effects on the pore open probability

Journal of Biological Chemistry, 1993

... side of the membrane, and that Ca2+ redistribution between the external inhibitory site and t... more

The Haves and Have-Nots: The Mitochondrial Permeability Transition Pore across Species

Cells

The demonstration that F1FO (F)-ATP synthase and adenine nucleotide translocase (ANT) can form Ca... more The demonstration that F1FO (F)-ATP synthase and adenine nucleotide translocase (ANT) can form Ca2+-activated, high-conductance channels in the inner membrane of mitochondria from a variety of eukaryotes led to renewed interest in the permeability transition (PT), a permeability increase mediated by the PT pore (PTP). The PT is a Ca2+-dependent permeability increase in the inner mitochondrial membrane whose function and underlying molecular mechanisms have challenged scientists for the last 70 years. Although most of our knowledge about the PTP comes from studies in mammals, recent data obtained in other species highlighted substantial differences that could be perhaps attributed to specific features of F-ATP synthase and/or ANT. Strikingly, the anoxia and salt-tolerant brine shrimp Artemia franciscana does not undergo a PT in spite of its ability to take up and store Ca2+ in mitochondria, and the anoxia-resistant Drosophila melanogaster displays a low-conductance, selective Ca2+-in...

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CyP-D modulates apoptosis triggered by detachment of HK II from mitochondria

<p>(A, B) Output of multiparametric FACS analyses show apoptosis induction in fibroblasts o... more <p>(A, B) Output of multiparametric FACS analyses show apoptosis induction in fibroblasts obtained either from wild-type mice (A) or from mice in which the <i>Ppif</i> gene encoding for CyP-D was ablated (B). Cells were exposed for 2 hours to the reported concentrations of TAT-HK with or without pre-incubation with Debio 025 (DB, 8 µM) or with a pan-caspase inhibitor (Z.VAD-fmk, 50 µM). Diagrams and percentages of the different cell populations are indicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001852#pone-0001852-g001" target="_blank">Fig. 1B</a>. All reported measures in the Figure are representative of at least four experiments.</p

Low P66shc with High SerpinB3 Levels Favors Necroptosis and Better Survival in Hepatocellular Carcinoma

Biology, 2021

Cell proliferation and escape from apoptosis are important pathological features of hepatocellula... more Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc−/− mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc−/− showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpr...

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Induction of the mitochondrial permeability transition by N-ethylmaleimide depends on secondary oxidation of critical thiol groups. Potentiation by copper-ortho-phenanthroline without dimerization of the adenine nucleotide translocase

Biochimica et biophysica acta, 1998

Addition to energized rat liver mitochondria of low micromolar concentrations of the thiol oxidan... more Addition to energized rat liver mitochondria of low micromolar concentrations of the thiol oxidant, copper-o-phenanthroline [Cu(OP)2], causes opening of the permeability transition pore, a cyclosporin A-sensitive channel. The effects of Cu(OP)2 can be reversed by reduction with dithiothreitol (DTT), suggesting that a dithiol-disulfide interconversion is involved. However, at variance with all pore inducers known to act through dithiol oxidation, the effects of Cu(OP)2 are not prevented by treatment of mitochondria with low (10-20 microM) concentrations of N-ethylmaleimide (NEM). Rather, these concentrations of NEM potentiate the inducing effects of Cu(OP)2. We show that this enhancing effect of NEM is blocked by the subsequent addition of DTT, indicating that potentiation by NEM is mediated by an oxidative event rather than by substitution as such. We find that also pore induction by high (0.5-1.0 mM) concentrations of NEM in the absence of oxidants is completely blocked by reductio...

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The Unique Cysteine of F-ATP Synthase OSCP Subunit Participates in Modulation of the Permeability Transition Pore

Cell Reports, 2020

Highlights d C141 of OSCP subunit of F-ATP synthase regulates the mitochondrial PTP d Oxidation o... more

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Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase dimers and to the generation of the full-conductance mitochondrial megachannel

Journal of Biological Chemistry, 2019

The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is... more The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the F O sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca 2؉-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.

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OSCP subunit of mitochondrial ATP synthase: role in regulation of enzyme function and of its transition to a pore

British Journal of Pharmacology, 2018

The permeability transition pore (PTP) is a latent, high-conductance channel of the inner mitocho... more The permeability transition pore (PTP) is a latent, high-conductance channel of the inner mitochondrial membrane. When activated, it plays a key role in cell death and therefore in several diseases. The pursuit of the PTP has taken an unexpected turn after the discovery that cyclophilin D (the target of the PTP inhibitory effect of cyclosporin A) binds F O F 1 (F)-ATP synthase resulting in inhibition of its catalytic activity by about 30% [Giorgio et al. (2009) J. Biol. Chem. 284, 33982-33988]. This observation was followed by the demonstration that binding occurs at the oligomycin sensitivity conferral protein (OSCP) and that F-ATP synthase can form Ca 2+-activated, high conductance channels with features matching those of the PTP, suggesting that the latter originates from a conformational change of F-ATP synthase [Giorgio et al. (2013) Proc Natl Acad Sci USA 110, 5887-5892]. This review is specifically dedicated to F-ATP synthase OSCP subunit, whose unique features make it a potential pharmacological target both for modulation of F-ATP synthase and its transition to a pore.

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Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter

Cardiovascular research, Jan 27, 2018

Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice abrogates mitochondrial Ca2+ upta... more Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice abrogates mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice are not protected from ischemic injury. We aimed to investigate whether adaptive alterations occur in cell death signaling pathways in the hearts of global MCU-KO mice. First, we examined whether cell death may occur via an upregulation in necroptosis in MCU-KO mice. However, our results show that neither RIP1 inhibition nor RIP3 knockout afford protection against ischemia-reperfusion injury in MCU-KO as in WT hearts, indicating that the lack of protection cannot be explained by upregulation of necroptosis. Instead, we have identified alterations in CypD signaling in MCU-KO hearts. In the presence of a calcium ionophore, MCU-KO mitochondria take up calcium and do undergo PTP opening. Furthermore, PTP opening in MCU-KO mitochondria has a lower calcium retention capacity, suggesting that the calcium ...

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Dopamine Oxidation Products as Mitochondrial Endotoxins, a Potential Molecular Mechanism for Preferential Neurodegeneration in Parkinson's Disease

ACS chemical neuroscience, Jan 3, 2018

The preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta is re... more The preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta is responsible for the motor impairment associated with Parkinson's disease. Dopamine is a highly reactive molecule, which is usually stored inside synaptic vesicles where it is stabilized by the ambient low pH. However, free cytosolic dopamine can auto-oxidize, generating reactive oxygen species, and lead to the formation of toxic quinones. In the present work, we have analyzed the mechanisms through which the dysfunction of dopamine homeostasis could induce cell toxicity, by focusing in particular on the damage induced by dopamine oxidation products at the mitochondrial level. Our results indicate that dopamine derivatives affect mitochondrial morphology and induce mitochondrial membrane depolarization, leading to a reduction of ATP synthesis. Moreover, our results suggest that opening of the mitochondrial transition pore induced by dopamine-derived quinones may contribute to the specif...

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Mitochondrial protein encoded in the X-II ORF of HTLV-I

Functional analysis of the p13II protein of HTLV-1

Functional analyses of P13II, a mitochondrial protein of human T-cell leukemia virus type-1

The P13 Protein of HTLV-1 Modulates Mitochondrial K plus Permeability, Reactive Oxygen Species and Cell Death

Differential proteome analysis of functionally different muscles in col6a1-/-mice vs controls in basal condition and after treatment with CsA

Clinical Trials, Databases and Registries; Poster Presen-Tations

Paolo Bernardi

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