PROGETTO DI RICERCA
Detailed biochemical analysis of a novel pathogenic cytochrome b mutation
in the cybrid cell model
Background
Ubiquinol:cytochrome c oxidoreductase (cytochrome bc1 or respiratory complex III, EC 1.10.2.2) is the central complex of the respiratory chain. In mammals, it comprises eleven subunits among which cytochrome b is the only one encoded by the mitochondrial genome[Vazquez-Acevedo et al. 1993]. Together with the iron-sulfur (Fe/S) protein and cytochrome c1 subunits, cytochrome b forms the catalytic core of complex III (CIII).In its native form, CIII is dimeric, being closely associated in varying proportions with complexes I (CI) and IV (CIV) to form the supercomplex CI+III+IV or respirasome [Schagger et al. 2000]. The respirasome confers structural and kinetic advantages being required for the assembly and stability of CI, favouring the substrate channelling and preventing excessive formation of reactive oxygen species [Schagger et al. 2004].
Mutations in the MTCYB gene, including deletions, frame-shifts, termination and missense mutations, are among the least common abnormalities identified to date in humans. Nevertheless, they exhibit a large spectrum of mitochondrial disease symptoms ranging from pure muscle symptoms, mostly exercise intolerance, to multisystem disorders, including some types of cancer [Benit et al., 2009].
Interestingly, it has been reported that some mutations inMTCYBare responsible for CI deficiency and mutations in CI genes induce CIII deficiency, underlining a close relationship between the two respiratory complexes [Acin-Perez et al. 2004]. MTCYBmutations can also increase reactive oxygen species (ROS) production, being indeed CIII and I the major ROS producers in mitochondria and in turn in cells [Murphy, 2009]. Recently, we proposed that the supramolecular organization of CI+III+IV can also be effective in mitigating the detrimental effects induced by the pathogenic mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C) in cytochrome b [Ghelli et al., 2013], highlighting the importance of maintaining SCs structure for respiratory complexes activity.
Aim
The aim of this project is to investigate the biochemical features of a novel pathogenic MTCYBmutation, the microdeletion (del15643-15660) of six aminoacids (I300 to P305) in the sixth transmembrane helix of the protein, identified in a 41-year-old woman suffering exercise intolerance and fatigability, sensorineural deafness, visual disturbances, postural and gait instability and dysphagia.
To this purpose transmitochondrial cytoplasmic hybrids (or cybrids) have been already developed, by fusing enucleated fibroblasts derived from the patient with osteosarcoma 143B.TK- cells deprived of their own mtDNA (Rho-0 cells) [King and Attardi 1996]. In particular both homoplasmic (100% mtDNA mutated) and heteroplasmic (containing both wild-type and mutated mtDNA) cybrid clones have been generated, in order to investigate the effect of the presence of a mixture of both wild type and mutated cytochrome b, which is the phenotype actually present in the patient’s tissues.
The final goal of the project is to better understand CIII-associated diseases for potential development of pharmacological treatments of affected patients.
Experimental plan
1. Determination of the oxidative phosphorylation efficiency of cybrids carrying the pathogenic MTCYB del15643-15660 microdeletion
We plan to carry out a panel of assays routinely performed in our laboratory in both homoplasmic and heteroplasmic (80% mutated mtDNA) cybrids, namely:
1.1. Determination of viability after metabolic stress When cells are grown in a glucose-free medium containing galactose (DMEM-galactose, supplemented with 5mM galactose, 5 mM sodium pyruvate, and 5%FBS), the rate of glycolysis is strongly reduced, and cells are forced to rely solely on oxidative phosphorylation to synthesize ATP. Under these conditions, cells bearing defects in the respiratory chain are unable to survive [Robinson et al. 1992; Ghelli et al. 2003]. Preliminary experiments showed that cybrids bearing the homoplasmic MTCYBmicrodeletion underwent a dramatic reduction of cell viability, determined by using the colorimetric sulforhodamine B assay, when incubated in DMEM-galactose.
1.2. Measurement of respiratory complexes activities
Enzymatic activity of CI (NADH:DB:DCIP), CII (Succ:DB:DCIP), CIII (DBH2:cytochrome c), CIV (reduced cytochrome c:O2), CI+III (NADH:cytochrome c) and CII+III (succinate:cytochrome c) will be determined in mitochondrial fractions isolated from wild type and mutant cybrids, as previously described [Ghelli et al., 2013]. Interestingly preliminary experiments revealed that CII activity was markedly increased in the homoplasmic mutant clone compared to control and heteroplasmic clones, prompting us to test whether this is associated with an increase in H2O2 overproduction, as recently reported by Acin-Perez et al., 2014.
1.3. Measurement of ROS production
To measure H2O2 overproduction, a standardized protocol employing the fluorescent dye dichlorofluorescein will be utilized. The cellular content of reduced and oxidized glutathione (GSH and GSSG) will then be determined, as previously described [Porcelli et al. 2008], given that increased GSSG/GSH ratio is considered a good indicator of the presence of an oxidative stress. Finally, the expression levels of the ROS scavenger Mn-SOD and catalase will be assessed, to reveal an imbalance in the antioxidant defences.
1.4. Effect of antioxidants on viability
If an increased production of H2O2 will be determined, we will then analyse whether pre-incubation with antioxidant compounds (N-acetylcysteine, tyron, etc) is able to counteract ROS overproduction and ameliorate the viability in galactose medium.
1.5. Rate of mitochondrial ATP production
Oxidative phosphorylation efficiency will be directly determined by measuring the rate of mitochondrial ATP synthesis in digitonin-permeabilized control and mutant cybrids using the luciferin/luciferase assay with a luminometer [Manfredi et al. 2002). ATP synthesis will be performed in the presence of malate and pyruvate (CII substrates), or succinate (CII substrate ) plus rotenone, or both CII and CII substrate (without rotenone). The rates of ATP synthesis will be normalized to the citrate synthase activity as an indication of mitochondrial mass content [Trounce et al. 1996]. This assay will allow to determine the overall mitochondrial oxidative phosphorylation capacity of mutant cells compared to controls.
2.Investigation of therespiratory supercomplexes
The expression levels of representative subunits of CIII as well as of other respiratory complexeswill be evaluated by immunoblot using commercially available antibodies. Then the steady-state levels of respiratory complexes and supercomplexes will be examined as cytochrome b mutants might lead to up-regulation of other respiratory complexes (in particular complex I and IV) [Acin-Perez et al. 2004; Schagger et al. 2004; D'Aurelio et al. 2006]. Mitochondria solubilized with DDM or digitonin will be analysed by usingBN-PAGE [D'Aurelio et al. 2006; Ghelli et al. 2013]. Preliminary results indicate that the SCs pattern is markedly different in homoplasmic and heteroplasmic clones compared with controls, suggesting that also this MTCTBmutation strongly affect the supramolecur organization of the respiratory complexes.
3.Analysis of respiratory supercomplexes under hypoxia and after treatment with antioxidants
Incubation under hypoxic conditions can alter the supramolecular organization of supercomplexes by inducing transcription of specific assembly factors. We will analyse whether the alteration of supercomplex I-III-IV organization observed in mitochondria bearing the microdeletion can be ameliorated after incubation under hypoxic conditions or after treatment with antioxidants.
This experiment will be also carried out in cybrids bearing the missense homoplasmic mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C) in cytochrome b, that has been deeply characterized in our lab [Ghelli et al. 2013; Lanciano et al, 2013].
References
Acin-Perez, R., M. P. Bayona-Bafaluy, et al. (2004). Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol Cell 13: 805-15.
Acin-Perez R, Carrascoso I, et al.( 2014) ROS-triggered phosphorylationof complex II by Fgr kinase regulatescellular adaptation to fuel use. Cell Metab 19, 1020–1033.
Angelini R, Vitale R, et al.(2012) Lipidomics of intact mitochondria by MALDI-TOF/MS.J Lipid Res53:1417-1425.
Bénit P., Lebon S., Rustin P. Respiratory-chain diseases related to complex III deficiency (2009)Biochim. Biophys. Acta 1793: 181–185.
Carelli, V., A. Achilli, et al. (2006) Haplogroup effects and recombination of mitochondrial DNA: novel clues from the analysis of Leber hereditary optic neuropathy pedigrees. Am J Hum Genet 78: 564-74.
D'Aurelio, M., C. D. Gajewski, et al. (2006) Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids. Hum Mol Genet 15: 2157-69.
DiMauro, S. and E. A. Schon (2008) Mitochondrial disorders in the nervous system. Annu Rev Neurosci 31: 91-123.
Fato, R., M. Cavazzoni, et al. (1993) Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects. Biochem J 290 ( Pt 1): 225-36.
Ghelli, A., C. Zanna, et al. (2003) "Leber's hereditary optic neuropathy (LHON) pathogenic mutations induce mitochondrial-dependent apoptotic death in transmitochondrial cells incubated with galactose medium." J Biol Chem 278: 4145-50.
Ghelli, A., A. M. Porcelli, et al. (2008) Protection against oxidant-induced apoptosis by exogenous glutathione in Leber hereditary optic neuropathy cybrids. Invest Ophthalmol Vis Sci 49: 671-6.
GhelliA, TropeanoCV, et al. (2013) The cytochrome b p.278Y>C mutation causative of a multisystem disorder enhances superoxide production and alters supramolecular interactions of respiratory chain complexes. Hum Mol Genet22: 2141-51.
Gohil VM, Hayes P, et al. (2004) Cardiolipin biosynthesis and mitochondrial respiratory chain function are interdependent.J Biol Chem 279:42612-8.
King, M. P. and G. Attardi (1996)Isolation of human cell lines lacking mitochondrial DNA. Methods Enzymol 264: 304-13.
LancianoP, Khalfaoui-HassaniB, et al. (2013) Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.Biochim Biophys Acta 1827: 1332-1339.
Lee, D.W., Selamoglu, N., et al. (2011) Loss of a conserved tyrosine residue of cytochrome b induces reactive oxygen species production by cytochrome bc1. J Biol Chem,286, 18139-48.
Manfredi, G., L. Yang, et al. (2002). Measurements of ATP in mammalian cells.Methods 26: 317-26.
Murphy M.P. (2009) How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13
Porcelli, A. M., A. Ghelli, et al. (2008) The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I. Cell Mol Life Sci 65: 2943-51.
Robinson, B. H., R. Petrova-Benedict, et al. (1992) Nonviability of cells with oxidative defects in galactose medium: a screening test for affected patient fibroblasts Biochem Med Metab Biol 48: 122-6.
Schagger H., Pfeiffer K. (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J.19: 1777–1783.
Schagger, H., R. de Coo, et al. (2004) Significance of respirasomes for the assembly/stability of human respiratory chain complex I. J Biol Chem 279(35): 36349-53.
Torroni, A., K. Huoponen, et al. (1996) Classification of European mtDNAs from an analysis of three European populations. Genetics 144(4): 1835-50.
Trounce, I. A., Y. L. Kim, et al. (1996) Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol 264: 484-509.
Vazquez-Acevedo M., Antaramian A., et al. (1993) Subunit structures of purified beef mitochondrial cytochrome bc1 complex from liver and heart. J. Bioenerg. Biomembr. 25: 401-410.
PIANO DELLE ATTIVITA’
Il piano di formazione associato a questo progetto prevede che il titolare dell’assegno approfondisca le conoscenze delle tecniche per l’analisi dei supercomplessi, effettuando diretta esperienza della tempistica di ri-assemblagio dei SC dopo inibizione della sintesi proteica mitocondriale. A tal fine si gioverà della collaborazione con la Dr.ssa Cristina Ugalde, Università di Madrid, esperta di questo tipo di analisi.
Specificamente il piano di formazione prevede che il candidato acquisisca esperienze riguardanti:
- Inibizione della sintesi proteica mitocondriale con specifici antibiotici e recovery;
- Separazione dei mitocondri a tempi diversi e analisi mediante BN-PAGE;
- Identificazione dei SCs mediante western blotting mediante anticorpi specifici.
- Eventuale analisi mediante 2D BN-PAGE/SDS PAGE.
Le competenze per queste tecniche per quanto riguarda in particolare le subunità che costituiscono il CIII sono disponibili nel dipartimento della Prof. Ugalde a Madrid, presso il cui laboratorio del titolare del progetto potrebbe recarsi per un breve periodo.
La formazione dell’assegnista prevede inoltre la partecipazione ai seminari del laboratorio e dipartimentali, Journal Club, congressi nazionali ed internazionali e scuole di specializzazione pertinenti e utili per lo svolgimento della ricerca.
Gruppo di Ricerca del Dipartimento in cui si inserirà l’assegnista
Prof. Giancarlo Solaini (Professore Associato Confermato)
Prof.ssa Michela Rugolo (Professore Associato Confermato)
Dott.ssa Anna Maria Ghelli (Ricercatore Universitario confermato)
Relazione con progetti di ricerca finanziati nel laboratorio
- Progetto di Ricerca di Interesse Nazionale (PRIN) 2013-2015. Mitochondrial mechanisms of cancerogenesis. Co-Investigator in a collaborative national project. (fonte del finanziamento)
- RFO 2013
Riassunto
Inglese -
The aim of this project is to investigate the biochemical features of a novel pathogenic MTCYB microdeletion (del15643-15660) of six aminoacids (I300 to P305) in the sixth transmembrane helix of the protein. The final goal of the project is to better understand the molecular alteration of complex III-associated diseases for potential development of pharmacological treatments of affected patients.
Italiano
Lo scopo del progetto è quello di analizzare le caratteristiche biochimichedella microdelezione (del15643-15660) di sei aminoacidi (da I300 a P305) nella sesta elica trans membrana della proteina. L’obiettivo finale è quello di comprendere meglio le alterazionimolecolari associate alle malattie del complesso III, per il futuro sviluppo di trattamenti farmacologici dei pazienti.
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