Electron and proton transfer in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

Cells of every living organism on our planet − bacterium, plant or animal − are organized in such a way that despite differences in structure and function they utilize the same metabolic energy represented by electrochemical proton gradient across a membrane. This gradient of protons is generated by the series of membrane bound multisubunit proteins, Complex I, II, III and IV, organized in so-called respiratory or electron transport chain. In the eukaryotic cell it locates in the inner mitochondrial membrane while in the bacterial cell it locates in the cytoplasmic membrane. The function of the respiratory chain is to accept electrons from NADH and ubiquinol and transfer them to oxygen resulting in the formation of water. The free energy released upon these redox reactions is converted by respiratory enzymes into an electrochemical proton gradient, which is used for synthesis of ATP as well as for many other energy dependent processes. This thesis is focused on studies of the first member of the respiratory chain − NADH:ubiquinone oxidoreductase or Complex I. This enzyme has a boot-shape structure with hydrophilic and hydrophobic domains, the former of which has all redox groups of the protein, the flavin and eight to nine iron-sulfur clusters. Complex I serves as a proton pump coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. So far the mechanism of energy transduction by Complex I is unknown. In the present study we applied a set of different methods to study the electron and proton transfer reactions in Complex I from Escherichia coli. The main achievement was the experiment that showed that the electron transfer through the hydrophilic domain of Complex I is unlikely to be coupled to proton transfer directly or to conformational changes in the protein. In this work for the first time properties of all redox centers of Complex I were characterized in the intact purified bacterial enzyme. We also probed the role of several conserved amino acid residues in the electron transfer of Complex I. Finally, we found that highly conserved amino acid residues in several membrane subunits form a common pattern with a very prominent feature – the presence of a few lysines within the membrane. Based on the experimental data, we suggested a tentative principle which may govern the redox-coupled proton pumping in Complex I.Kaikkien planeetallamme elollisten olentojen – bakteerien, kasvien sekä eläinten – solut käyttävät samaa metabolisen energian perusmuotoa, kalvon yli muodostettavaa elektrokemiallista protonigradienttia. Tämän protonigradientin saa aikaan sarja monista alayksiköistä koostuvia kalvoproteiineja (kompleksit I, II, III ja IV), jotka ovat järjestäytyneet niin sanotuksi hengitys- tai elektroninsiirtoketjuksi. Eukaryoottisolussa entsyymit sijaitsevat mitokondrion sisemmässä kalvossa, kun taas bakteereissa ne löytyvät solukalvosta. Hengitysketjun tarkoitus on vastaanottaa elektroneita NADH:lta ja ubikinolilta sekä siirtää ne edelleen happelle muodostaen samalla vettä. Hengitysketjun entsyymit muuntavat näissä hapetus-pelkistysreaktioista vapautuvan energian sähkökemialliseksi protonigradientiksi, jota käytetään ATP synteesissä, ja myös lukuisissa muissa energiaa vaativissa prosesseissa. Tämä väitöskirja keskittyy hengitysketjun ensimmäisen entsyymin, NADH:ubikonini oksidoreduktaasin eli kompleksi I:n, tutkimiseen. Tällä entsyymillä on L-kirjaimen muotoinen rakenne, jossa vesiliukoinen osa on kiinnittyneenä kalvossa sijaitsevan osan toiseen päähän. Hydrofiilisessä osassa sijaitsevat entsyymin kaikki hapetus-pelkistyskeskukset, flaviinimolekyyli ja kahdeksasta yhdeksään rauta-rikkikeskusta. Kompleksi I toimii protonipumppuna yhdistäen kahden elektronin siirron NADH:lta ubikinonille neljän protonin pumppaamiseen kalvon yli. Toistaiseksi kompleksi I:n toimintamekanismi on vielä tuntematon. Tässä tutkimuksessa käytimme erilaisten menetelmien yhdistelmää tutkiaksemme elektronin- ja protoninsiirto reaktioita Escherichia coli:sta eristetyssä kompleksi I:ssä. Keskeisin saavutus oli osoitus, että elektroninsiirto kompleksi I:n hydrofiilisen domeeni läpi ei näytä kytkeytyvän suoraan eikä välillisesti konformaation muutoksen avulla protoninsiirtoon. Ensimmäistä kertaa kaikkien kompleksi I:n hapetus-pelkistyskeskusten ominaisuudet kartoitettiin bakteerista eristetyssä entsyymissä. Tutkimme myös useiden vakioisten aminohappotähteiden merkitystä kompleksi I:n elektroninsiirrossa. Lisäksi havaitsimme vakioisten aminohappojen muodostaman toistuvat mallin useissa kalvon läpäisevissä alayksiköissä. Keskeisin havainto mallissa on kalvon sisällä sijaitsevat lysiini aminohapot. Kokeellisten tulosten perusteella esitimme hypoteesin kompleksi I:n toimintaperiaatteesta

Simultaneous measurement of folate cycle intermediates in different biological matrices using liquid chromatography-tandem mass spectrometry

The folate cycle is an essential metabolic pathway in the cell, involved in nucleotide synthesis, maintenance of the redox balance in the cell, methionine metabolism and re-methylation reactions. Standardised methods for the measurement of folate cycle intermediates in different biological matrices are in great demand. Here we describe a rapid, sensitive, precise and accurate liquid chromatographic-tandem mass spectrometric (LC-MS/ MS) method with a wide calibration curve range and a short run time for the simultaneous determination of folate cycle metabolites, including tetrahydrofolic acid (THF), 5-methyl THF, 5-formyl THF, 5,10-methenyl THF, 5,10-methylene THF, dihydrofolic acid (DHF) and folic acid in different biological matrices. Extraction of folate derivatives from soft and hard tissue samples as well as from adherent cells was achieved using homogenisation in buffer, while extraction from the whole blood and plasma relied on the anion exchange solid-phase extraction (SPE) method. Chromatographic separation was completed using a Waters Atlantis dC(18) 2.0 x 100 mm, 3-mu column with a gradient elution using formic acid in water (0.1% v/v) and acetonitrile as the mobile phases. LC gradient started with 95% of the aqueous phase which was gradually changed to 95% of the organic phase during 2.70 min in order to separate the selected metabolites. The analytes were separated with a run time of 5 min at a flow rate of 0.300 mL/min and detected using a Waters Xevo-TQS triple quadrupole mass spectrometer in the multiple reaction monitoring mode (MRM) at positive polarity. The instrument response was linear over a calibration range of 0.5 to 2500ng/mL (r(2) > 0.980). The developed bioanalytical method was thoroughly validated in terms of accuracy, precision, linearity, recovery, sensitivity and stability for tissue and blood samples. The matrix effect was compensated by using structurally similar isotope labelled internal standard (IS), C-13(5)-methyl THF, for all folate metabolites. However, not all folate metabolites can be accurately quantified using this method due to their high interconversion rates especially at low pH. This applies to 5,10-methylene THF which interconverts into THF, and 5,10-methenyl-THF interconverting into 5-formyl-THF. Using this method, we measured folate cycle intermediates in mouse bone marrow cells and plasma, in human whole blood; in mouse muscle, liver, heart and brain samples.Peer reviewe

Atomistic Molecular Dynamics Simulations of Mitochondrial DNA Polymerase gamma : Novel Mechanisms of Function and Pathogenesis

DNA polymerase gamma (Pol gamma) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol gamma replicative complex. Our simulation data show that DNA binding triggers remarkable changes in the enzyme structure, including (1) completion of the DNA-binding channel via a dynamic subdomain, which in the apo form blocks the catalytic site, (2) stabilization of the structure through the distal accessory beta-subunit, and (3) formation of a putative transient replisome-binding platform in the "intrinsic processivity" subdomain of the enzyme. Our data indicate that noncatalytic mutations may disrupt replisomal interactions, thereby causing Pol gamma-associated neurodegenerative disorders.Peer reviewe

Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of hte human mitochondrial replicase, Pol gamma

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Loss of mtDNA activates astrocytes and leads to spongiotic encephalopathy

Mitochondrial dysfunction manifests as different neurological diseases, but the mechanisms underlying the clinical variability remain poorly understood. To clarify whether different brain cells have differential sensitivity to mitochondrial dysfunction, we induced mitochondrial DNA (mtDNA) depletion in either neurons or astrocytes of mice, by inactivating Twinkle (TwKO), the replicative mtDNA helicase. Here we show that astrocytes, the most abundant cerebral cell type, are chronically activated upon mtDNA loss, leading to early-onset spongiotic degeneration of brain parenchyma, microgliosis and secondary neurodegeneration. Neuronal mtDNA loss does not, however, cause symptoms until 8 months of age. Findings in astrocyte-TwKO mimic neuropathology of Alpers syndrome, infantile-onset mitochondrial spongiotic encephalopathy caused by mtDNA maintenance defects. Our evidence indicates that (1) astrocytes are dependent on mtDNA integrity; (2) mitochondrial metabolism contributes to their activation; (3) chronic astrocyte activation has devastating consequences, underlying spongiotic encephalopathy; and that (4) astrocytes are a potential target for interventions.Peer reviewe

Redox regulation of GRPEL2 nucleotide exchange factor for mitochondrial HSP70 chaperone

Mitochondria are central organelles to cellular metabolism. Their function relies largely on nuclear-encoded proteins that must be imported from the cytosol, and thus the protein import pathways are important for the maintenance of mitochondrial proteostasis. Mitochondrial HSP70 (mtHsp70) is a key component in facilitating the translocation of proteins through the inner membrane into the mitochondrial matrix. Its protein folding cycle is regulated by the nucleotide-exchange factor GrpE, which triggers the release of folded proteins by ATP rebinding. Vertebrates have two mitochondrial GrpE paralogs, GRPEL1 and 2, but without clearly defined roles. Using BioID proximity labeling to identify potential binding partners of the GRPELs in the mitochondrial matrix, we obtained results supporting a model where both GRPELs regulate mtHsp70 as homodimers. We show that GRPEL2 is not essential in human cultured cells, and its absence does not prevent mitochondrial protein import. Instead we find that GRPEL2 is redox regulated in oxidative stress. In the presence of hydrogen peroxide, GRPEL2 forms dimers through intermolecular disulfide bonds in which Cys87 is the thiol switch. We propose that the dimerization of GRPEL2 may activate the folding machinery responsible for protein import into mitochondrial matrix or enhance the chaperone activity of mtHSP70, thus protecting mitochondrial proteostasis in oxidative stress.Peer reviewe

Lihaskudoksen NAD+-puutos ja Serpina3n molekyylibiologisina tekijöinä hiirten syöpäkakeksiassa – myostatiinin ja aktiviinien estäjien vaikutus

Syöpäkakeksia on patofysiologialtaan vielä suurelta osin tuntematon, monitekijäinen aineenvaihdunnallinen sairaus, jossa paino ja lihaskudoksen määrä laskevat. Kakeksia vaikuttaa suuren osaan syöpäpotilaista lisäten heidän kuolleisuuttaan ja heikentäen elämänlaatua. Eläinmalleilla lihaskadon estäminen kokeellisella hoidolla, aktiviinireseptorin salpauksella, on parantanut selviytymistä ja vähentänyt lihasmassan katoa, vaikka syöpä itsessään ei ole parantunut. Tutkimuksemme tavoitteena oli selvittää tämän hoidon vaikutusmekanismi, sillä se on tällä hetkellä vielä epäselvää. Tutkimuksessa hiirille injektoitiin suolistosyöpäsoluja (C26) selän rasvakudokseen, mikä aiheuttaa pahanlaatuisen kasvaimen muodostumisen ja nopean lihaskadon. Hiirille annettiin kokeellista hoitoa yhdisteellä, joka estää lihasmassan kasvua rajoittavien proteiinien, aktiviinien ja myostatiinien, kiinnittymistä niiden solureseptoreihin. Havaitsimme laajoissa proteiinianalyyseissa akuutin faasin reaktion, erityisesti Serpina3n-proteiinin, lisääntymisen ja lihaksen mitokondrioiden energiaa tuottavan oksidatiivisen fosforylaation (OXPHOS) komponenttien määrän vähentyneen syöpäryhmässä. Niinpä analysoimme tarkemmin mitokondrioiden aktiivisuutta histokemiallisesti in situ ja määritimme mitokondrioiden energiantuotantoreaktioissa toimivan elintärkeän kofaktorin, nikotiiniamidiadeniinidinukleotidin (NAD+), pitoisuuksia lihaskudoksessa. Mitokondrioaktiivisuus in situ oli osittain heikentynyt syöpäryhmässä, jossa myös NAD+:n ja sen metaboliitin NADH:n määrät vähentyivät. Lisäksi havaitsimme voimakkaan laskun NAD+-biosynteesiin osallistuvan geenin, Nrk2:den ilmaantumisessa. Proteiineista Serpina3n määrä korreloi selkeimmin painonlaskuun, mutta se paljastui ennemmin hoitamattoman kakeksian kuin yleisen lihasmassan biomarkkeriksi. Serpina3n-määrä yhdistyi myös Nrk2:den ilmentymiseen, joka viittaisi yhteyteen lihaksen Nrk2-ekspressiossa ja tulehduksen säätelyssä akuutin faasin reaktion kautta. Aktiviinireseptorin salpauksella pystyimme korjaamaan NAD+-aineenvaihdunnan häiriötä ja lieventämään OXPHOS-muutoksia. Osoitimme tässä tutkimuksessa ensimmäistä kertaa, että syöpäkakeksian taustalla on NAD+-aineenvaihdunnan häiriö ja että aktiviinireseptorien salpauksella voidaan lihaskadon estämisen lisäksi parantaa NAD+-tasoja. Vielä ei tiedetä, vaikuttaako tämä kokeellinen hoito suoraan vai välillisesti NAD+-aineenvaihduntaan. Tulevaisuudessa olisi lisäksi tärkeä tutkia Nrk2:den roolia syöpäkakeksiassa ja selvittää, voidaanko NAD+-esiasteita käyttää syöpäkakeksian hoitona

Muscle NAD+ depletion and Serpina3n as molecular determinants of murine cancer cachexia – the effects of blocking myostatin and activins

Objective Cancer cachexia and muscle loss are associated with increased morbidity and mortality. In preclinical animal models, blocking activin receptor (ACVR) ligands has improved survival and prevented muscle wasting in cancer cachexia without an effect on tumour growth. However, the underlying mechanisms are poorly understood. The present study aimed to identify cancer cachexia and soluble ACVR (sACVR) administration-evoked changes in muscle proteome. Methods Healthy and C26 tumour-bearing (TB) mice were treated with recombinant sACVR. The sACVR or PBS control were administered either prior to the tumour formation or by continued administration before and after tumour formation. Muscles were analysed by quantitative proteomics with further examination of mitochondria and nicotinamide adenine dinucleotide (NAD+) metabolism. To complement the first prophylactic experiment, sACVR (or PBS) was injected as a treatment following tumour cell inoculation. Results Muscle proteomics in TB cachectic mice revealed downregulated signatures for mitochondrial oxidative phosphorylation (OXPHOS) and increased acute phase response (APR). These were accompanied by muscle NAD+ deficiency, alterations in NAD+ biosynthesis including downregulation of nicotinamide riboside kinase 2 (Nrk2), and decreased muscle protein synthesis. The disturbances in NAD+ metabolism and protein synthesis were rescued upontreatment with sACVR. Across the whole proteome and APR in particular, Serpina3n represented the most upregulated protein and the strongest predictor of cachexia. However, the increase in Serpina3n expression associated with increased inflammation rather than decreased muscle mass and/or protein synthesis. Conclusions We present here an evidence implicating disturbed muscle mitochondrial OXPHOS proteome and NAD+ homeostasis in experimental cancer cachexia. Treatment of tumour-bearing mice with a blocker of activin receptor ligands restores depleted muscle NAD+ and Nrk2 as well as decreased muscle protein synthesis. These results point out putative new treatment therapies for cachexia. Our results also reveal that although acute phase protein Serpina3n may serve as a predictor of cachexia, it more likely reflects a condition of elevated inflammation.Peer reviewe

ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.Peer reviewe

Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3

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