The unique histidine of F-ATP synthase subunit OSCP mediates regulation of the permeability transition by matrix pH

The “Permeability transition” (PT) is one of the most studied events that may trigger cell death and is due to a Ca2+- and ROS-dependent opening of a nonspecific pore, called PTP, whose molecular nature has been long debated. Recently, our research group has demonstrated that PTP forms from FoF1 ATP synthase dimers, demonstrating the ability of this complex to switch from the key enzyme for the aerobic synthesis of ATP into a potential cell death mediator. The goal of this PhD thesis has been to define which structural changes of ATP synthase are responsible for the pH modulation of PTP. Indeed, it is well known from nineties that the optimum matrix pH for PT occurrence is about 7.3, and a decrease leads to decreased probability of PTP opening. The pH effect has been ascribed to reversible protonation of His residues located on the PTP that can be blocked by the histidine modifying reagent diethyl pyrocarbonate (DPC). Moreover, in mammalian cells, similarly to the drug Cyclosporin A (CsA), acidic pH also promotes release from the inner membrane of the matrix protein Cyclophilin D (CyPD), which is a well-known PTP activator. As our group demonstrated that CyPD binds to the ATP Synthase OSCP subunit, mainly through electrostatic interactions and resulting in partial enzyme inhibition, the hypothesis has been advanced that the unique histidine located on OSCP, His112 according to bovine numbering, may be responsible for both the pH effects on CyPD (un)binding to ATP synthase and on PTP/ATP synthase opening. OSCPHis112 is exposed to the solvent and is located in the flexible linker region between the structured N- and C-terminal domains of OSCP. The results obtained by ATP synthase immunoprecipitation from bovine heart mitochondria showed that acidic pH induces CyPD release that is prevented by DPC, perfectly matching the effect of DPC on CyPD-PTP interaction. DPC also prevented the binding at low pH of the inhibitor protein IF1 to ATP synthase, but this effect is probably not relevant to PTP modulation. ESI-MS and ESI-MS/MS analyses of the OSCP isolated from DPC-treated mitochondria revealed that the 95-113 peptide shows a mass shift of +72 Da, which is indicative of carbethoxylation of the unique His112. These data therefore strongly support the hypothesis that OSCP His112 is part of the binding site of CyPD on the protein, so that its protonation by lowering pH favors CyPD release. Of note, this region contains several residues of glutamic acid conferring a low potential surface, which is complementary to the mainly high potential surface of CyPD. Consistently to this model, DPC inhibits the ATPase activity of ATP synthase only when CyPD is released from OSCP, i.e. in the presence of CsA and in mitochondria from CyPD-null mice. Replacement of OSCPHis112 with a Gln in HEK cells, by the CRISPR/Cas9 system, showed its involvement even in the effect of low pH on PTP opening. Indeed, the PTP open probability is not affected by acidic pH only in mutated cells, while DPC reverts the pH inhibition exclusively in wild type cells. Finally, evaluation of the structural stability of the ATP Synthase dimers at low pH by Blue-native PAGE excluded their destabilization, which could affect PTP formation. In summary, these data provide a convincing model for the pH modulation of PTP, as well as a compelling evidence that ATP synthase and PTP are the same molecular entity

Congenital myopathy with hanging big toe due to homozygous myopalladin (MYPN) mutation

Background: Myopalladin (MYPN) is a component of the sarcomere that tethers nebulin in skeletal muscle and nebulette in cardiac muscle to alpha-actinin at the Z lines. Autosomal dominant MYPN mutations cause hypertrophic, dilated, or restrictive cardiomyopathy. Autosomal recessive MYPN mutations have been reported in only six families showing a mildly progressive nemaline or cap myopathy with cardiomyopathy in some patients. Case presentation: A consanguineous family with congenital to adult-onset muscle weakness and hanging big toe was reported. Muscle biopsy showed minimal changes with internal nuclei, type 1 fiber predominance, and ultrastructural defects of Z line. Muscle CT imaging showed marked hypodensity of the sartorius bilaterally and MRI scattered abnormal high-intensity areas in the internal tongue muscle and in the posterior cervical muscles. Cardiac involvement was demonstrated by magnetic resonance imaging and late gadolinium enhancement. Whole exome sequencing analysis identified a homozygous loss of function single nucleotide deletion in the exon 11 of the MYPN gene in two siblings. Full-length MYPN protein was undetectable on immunoblotting, and on immunofluorescence, its localization at the Z line was missed. Conclusions: This report extends the phenotypic spectrum of recessive MYPN-related myopathies showing: (1) the two patients had hanging big toe and the oldest one developed spine and hand contractures, none of these signs observed in the previously reported patients, (2) specific ultrastructural changes consisting in Z line fragmentation, but (3) no nemaline or caps on muscle pathology

The unique histidine of F-ATP synthase subunit OSCP mediates regulation of the permeability transition by matrix pH

The “Permeability transition” (PT) is one of the most studied events that may trigger cell death and is due to a Ca2+- and ROS-dependent opening of a nonspecific pore, called PTP, whose molecular nature has been long debated. Recently, our research group has demonstrated that PTP forms from FoF1 ATP synthase dimers, demonstrating the ability of this complex to switch from the key enzyme for the aerobic synthesis of ATP into a potential cell death mediator. The goal of this PhD thesis has been to define which structural changes of ATP synthase are responsible for the pH modulation of PTP. Indeed, it is well known from nineties that the optimum matrix pH for PT occurrence is about 7.3, and a decrease leads to decreased probability of PTP opening. The pH effect has been ascribed to reversible protonation of His residues located on the PTP that can be blocked by the histidine modifying reagent diethyl pyrocarbonate (DPC). Moreover, in mammalian cells, similarly to the drug Cyclosporin A (CsA), acidic pH also promotes release from the inner membrane of the matrix protein Cyclophilin D (CyPD), which is a well-known PTP activator. As our group demonstrated that CyPD binds to the ATP Synthase OSCP subunit, mainly through electrostatic interactions and resulting in partial enzyme inhibition, the hypothesis has been advanced that the unique histidine located on OSCP, His112 according to bovine numbering, may be responsible for both the pH effects on CyPD (un)binding to ATP synthase and on PTP/ATP synthase opening. OSCPHis112 is exposed to the solvent and is located in the flexible linker region between the structured N- and C-terminal domains of OSCP. The results obtained by ATP synthase immunoprecipitation from bovine heart mitochondria showed that acidic pH induces CyPD release that is prevented by DPC, perfectly matching the effect of DPC on CyPD-PTP interaction. DPC also prevented the binding at low pH of the inhibitor protein IF1 to ATP synthase, but this effect is probably not relevant to PTP modulation. ESI-MS and ESI-MS/MS analyses of the OSCP isolated from DPC-treated mitochondria revealed that the 95-113 peptide shows a mass shift of +72 Da, which is indicative of carbethoxylation of the unique His112. These data therefore strongly support the hypothesis that OSCP His112 is part of the binding site of CyPD on the protein, so that its protonation by lowering pH favors CyPD release. Of note, this region contains several residues of glutamic acid conferring a low potential surface, which is complementary to the mainly high potential surface of CyPD. Consistently to this model, DPC inhibits the ATPase activity of ATP synthase only when CyPD is released from OSCP, i.e. in the presence of CsA and in mitochondria from CyPD-null mice. Replacement of OSCPHis112 with a Gln in HEK cells, by the CRISPR/Cas9 system, showed its involvement even in the effect of low pH on PTP opening. Indeed, the PTP open probability is not affected by acidic pH only in mutated cells, while DPC reverts the pH inhibition exclusively in wild type cells. Finally, evaluation of the structural stability of the ATP Synthase dimers at low pH by Blue-native PAGE excluded their destabilization, which could affect PTP formation. In summary, these data provide a convincing model for the pH modulation of PTP, as well as a compelling evidence that ATP synthase and PTP are the same molecular entity.La transizione di permeabilità (PT) mitocondriale è uno degli eventi coinvolti nella morte cellulare tra i più studiati e implica l’apertura nella membrana mitocondriale interna di un poro aspecifico, definito PTP, indotta da ioni calcio in presenza di stress ossidativo, la cui natura molecolare è stata a lungo dibattuta. Recentemente, il gruppo di ricerca presso cui è stata svolta la presente tesi di dottorato ha dimostrato che il PTP si forma da complessi dimerici dell’enzima FoF1 ATP Sintetasi, trasformando l’enzima essenziale per la sintesi aerobica di ATP in mediatore di morte cellulare. L’obiettivo della presente tesi è stato quello di definire le basi molecolari della modulazione del PTP da parte del pH di matrice rispetto alle proprietà strutturali di ATP Sintetasi. E’ infatti noto dagli anni ’90 che le probabilità di apertura del PTP sono massime a pH 7.3 e diminuiscono rapidamente al diminuire del pH. Tale effetto è stato imputato alla protonazione di residui di istidine del PTP, in quanto l’inibizione veniva bloccata dal reagente specifico per le istidine dietilpirocarbonato (DPC). Inoltre, era stato dimostrato che in cellule di mammifero un pH acido favorisce, similmente alla Ciclosporina A (CsA), il rilascio dal PTP della proteina di matrice Ciclofilina D (CyPD), che, quando legata al PTP, è un ben noto attivatore del poro. Poiché studi recenti dello stesso gruppo avevano dimostrato che in mammifero la ciclofilina D interagisce, prevalentemente mediante interazioni elettrostatiche, con la subunità OSCP di ATP Sintetasi, inducendo un’inibizione parziale dell’attività catalitica dell’enzima, si è ipotizzato che l’unico residuo di istidina di OSCP, His112 in base alla numerazione in bovino, fosse coinvolto nella modulazione pH-dipendente sia del legame della CyPD che della probabilità di apertura del PTP/ATP Sintetasi. OSCPHis112 è esposto al solvente ed è localizzato nella parte non strutturata della proteina, legante i domini ad α-elica N- e C-terminali. I risultati ottenuti mediante immunoprecipitazione di ATP Sintetasi da mitocondri di cuore bovino hanno dimostrato che pH acidi inducono il rilascio della CyP e che tale rilascio viene bloccato dalla presenza di DPC, in perfetto accordo con quanto precedentemente osservato rispetto al PTP. L’analisi ESI-MS e ESI-MS/MS della subunità OSCP isolata da mitocondri trattati con DPC ha stabilito che il peptide 95-113 di OSCP subisce un aumento della sua massa molecolare corrispondente a 72 Da, indicativo della carbetossilazione dell’unica istidina. Questi dati suggeriscono quindi che OSCPHis112 sia coinvolta nel sito di legame della CyPD, in quanto l’interazione viene sfavorita dalla sua protonazione. His112 è infatti localizzato in una regione caratterizzata dalla presenza di numerosi residui di acido glutammico avente un potenziale di superficie negativo, che è complementare al potenziale di superficie della CyPD prevalentemente positivo. In accordo con questo modello, il DPC è risultato un inibitore dell’attività catalitica di ATP Sintetasi esclusivamente quando la CyPD non è legata ad OSCP, come in presenza di CsA o nei topi KO per la CyPD (Ppif-/-). Inoltre, la sostituzione di OSCPHis112 con un residuo di Gln mediante il sistema di mutagenesi CRISPR/Cas9 in una linea cellulare umana (HEK293T) ha permesso di verificare che la stessa istidina è coinvolta nell’effetto del pH sull’apertura del PTP, in quanto solo nelle cellule mutate la probabilità di apertura del PTP non diminuisce a pH acido, e, in accordo, il DPC reverte l’inibizione a pH acido esclusivamente nelle cellule wild type. Infine, l’analisi della stabilità strutturale dei dimeri di ATP Sintetasi in funzione del pH mediante Blue-native PAGE ha consentito di escludere una loro destabilizzazione a pH acidi, che avrebbe potuto influire sulla formazione del PTP. In conclusione, questi risultati, oltre a fornire un modello convincente per la modulazione pH-dipendente del PTP, costituiscono altresì una prova molecolare importante che ATP Sintetasi e il PTP sono la stessa entità molecolare

The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP Synthase: Emerging New Roles in Mitochondrial Pathophysiology

The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial FOF1 ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F1 sector, it makes stable contacts with both F1 and the peripheral stalk, ensuring the structural and functional coupling between FO and F1, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca2+ required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca2+-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death

Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients

The unique histidine in OSCP subunit of F-ATP synthase mediates inhibition of the permeability transition pore by acidic pH

The permeability transition pore (PTP) is a Ca2+-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 H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (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 harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F-ATP synthase