Paolo Bernardi - Academia.edu (original) (raw)

Papers by Paolo Bernardi

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...

The FEBS Journal, 2021

Major progress has been made in defining the basis of the mitochondrial permeability transition, ... more Major progress has been made in defining the basis of the mitochondrial permeability transition, a Ca2+‐dependent permeability increase of the inner membrane that has puzzled mitochondrial research for almost 70 years. Initially considered an artefact of limited biological interest by most, over the years the permeability transition has raised to the status of regulator of mitochondrial ion homeostasis and of druggable effector mechanism of cell death. The permeability transition is mediated by opening of channel(s) modulated by matrix cyclophilin D, the permeability transition pore(s) (PTP). The field has received new impulse (a) from the hypothesis that the PTP may originate from a Ca2+‐dependent conformational change of F‐ATP synthase and (b) from the reevaluation of the long‐standing hypothesis that it originates from the adenine nucleotide translocator (ANT). Here, we provide a synthetic account of the structure of ANT and F‐ATP synthase to discuss potential and controversial m...

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 ...

Cell Calcium, 2016

Mitochondria-dependent programmed cell death (PCD) in yeast shares many features with the intrins... more Mitochondria-dependent programmed cell death (PCD) in yeast shares many features with the intrinsic apoptotic pathway of mammals. With many stimuli, increased cytosolic [Ca 2+ ] and ROS generation are the triggering signals that lead to mitochondrial permeabilization and release of proapoptotic factors, which initiates yeast PCD. While in mammals the permeability transition pore (PTP), a high-conductance inner membrane channel activated by increased matrix Ca 2+ and oxidative stress, is recognized as part of this signaling cascade, whether a similar process occurs in yeast is still debated. The potential role of the PTP in yeast PCD has generally been overlooked because yeast mitochondria lack the Ca 2+ uniporter, which in mammals allows rapid equilibration of cytosolic Ca 2+ with the matrix. In this short review we discuss the nature of the yeast permeability transition and reevaluate its potential role in the effector phase of yeast PCD triggered by Ca 2+ and oxidative stress.

<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

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...

Nature Communications, 2019

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP),... more The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+dissipates the H+gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+can transform ...

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.

FEBS Letters, 1985

This paper analyzes the kinetics of the Caz+ uniporter of mitochondria from rat heart, kidney and... more This paper analyzes the kinetics of the Caz+ uniporter of mitochondria from rat heart, kidney and liver operating in a range of Caz+ concentrations near the steady-state value (l-4 PM). Heart mitochondria exhibit the lowest activity, and physiological Mgz+ concentrations inhibit the mitochondrial Caz+ uniporter by approx. 50% in heart and kidney, and by 20% in liver. At physiological Caz+ and Mg2+ concentrations the external free Ca2+ maintained by respiring mitochondria in vitro is higher in heart and kidney with respect to liver mitochondria. This behaviour could represent an adaptation of different mitochondria to their specific intracellular environment.

Biophysical Journal, 1999

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996

The permeability transition pore (MTP) is a high conductance channel of the mitochondrial inner m... more The permeability transition pore (MTP) is a high conductance channel of the mitochondrial inner membrane inhibited by cyclosporin A. While the physiological role of the MTP has not been clarified yet, it is becoming clear that this channel plays an important role in the pathways leading to cell death. The recent demonstrations that the MTP is controlled by the membrane potential, that a variety of physiological and pathological effectors can modulate the threshold voltage at which pore opening occurs, and that surface potential may contribute to pore modulation provide a useful framework to describe the mechanistic aspects of pore function in isolated mitochondria. Here we (i) briefly review the key features of pore regulation, and report our recent progress on the role of oxidants and mitochondrial cyclophilin; and (ii) elaborate on how MTP regulation by cellular pathophysiological effectors (such as cytosolic [Ca 2.] transients, oxidative stress, and changes in the concentration of polyamines, nitric oxide, and metabolites of both the sphingomyelin and phospholipase A 2 pathways) might take place in vivo. Further definition of the MTP checkpoints should help in the design of specific modulators, and offers great promise for the development of new conceptual and pharmacological tools aimed at therapeutic intervention in pathological conditions where pore opening is a critical event.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008

European Journal of Anaesthesiology, 2000

... Scorrano, Luca; Petronilli, Valeria; Penzo, Daniele; Bernardi, Paolo*Section Editor(s): De De... more ... Scorrano, Luca; Petronilli, Valeria; Penzo, Daniele; Bernardi, Paolo*Section Editor(s): De Deyne, C.; De Jongh, R.; Heylen, R.; Van Aken, H. Article Outline. Collapse Box Author Information. ... 4 Wissing D, Mouritzen H, Egeblad M, Poirier GG, Jaattela M. Involvement of caspase ...

Myosclerosis are diseases caused bymutations in the genes encoding the extracellularmatrix protei... more Myosclerosis are diseases caused bymutations in the genes encoding the extracellularmatrix protein collagen VI. A dystrophic mouse model, where collagen VI synthesis was prevented by targeted inactivation of theCol6a1 gene, allowed the investigation of pathogenesis,which revealed the existence of a Ca2þ-mediated dysfunction of mitochondria and sarcoplasmic reticulum, and of defective autophagy. Key events are dysregulation of the mitochondrial permeability transition pore, an inner membrane high-conductance channel that for pro-longed open times causes mitochondrial dysfunction, and inadequate removal of defective mitochondria, which amplifies the damage. Consistently, the Col6a12/2 myopathic mice could be cured through inhibition of cyclophilinD, amatrix protein that sensitizes the pore to opening, and through stimulation of autophagy. Similar defects contribute to disease path-ogenesis in patients irrespective of the genetic lesion causing the collagen VI defect. These studies ind...

Nature Communications

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but... more F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.

Cell Reports

Highlights d C141 of OSCP subunit of F-ATP synthase regulates the mitochondrial PTP d Oxidation o... more Highlights d C141 of OSCP subunit of F-ATP synthase regulates the mitochondrial PTP d Oxidation of OSCP C141 sensitizes the PTP to the inducing effect of diamide d CyPD exerts a protecting effect by masking OSCP C141 d Genetic ablation of CyPD sensitizes PTP to diamide

Biochimica et biophysica acta, Jan 22, 2018

Idebenone is a hydrophilic short-chain coenzyme (Co) Q analogue, which has been used as a potenti... more Idebenone is a hydrophilic short-chain coenzyme (Co) Q analogue, which has been used as a potential bypass of defective complex I in both Leber Hereditary Optic Neuropathy and OPA1-dependent Dominant Optic Atrophy. Based on its potential antioxidant effects, it has also been tested in degenerative disorders such as Friedreich's ataxia, Huntington's and Alzheimer's diseases. Idebenone is rapidly modified but the biological effects of its metabolites have been characterized only partially. Here we have studied the effects of quinones generated during in vivo metabolism of idebenone with specific emphasis on 6-(9-carboxynonyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (QS10). QS10 partially restored respiration in cells deficient of complex I or of CoQ without inducing the mitochondrial permeability transition, a detrimental effect of idebenone that may offset its potential benefits [Giorgio et al. (2012) Biochim. Biophys. Acta 1817: 363-369]. Remarkably, respiration was larg...

EMBO reports, Feb 1, 2018

The permeability transition pore (PTP) is a Ca-dependent mitochondrial channel whose opening caus... more The permeability transition pore (PTP) is a Ca-dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by His mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial FF(F)-ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP-dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch-clamp recordings of mitoplasts. Cells harbori...