RESUMO
Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4-/- mouse, to define the structural, biochemical, and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of the complex I from ndufs4-/- mouse hearts revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12, which replaces its paralogue NDUFAF2 in mature complex I, is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free enzyme. We propose that NDUFAF2 recruits the NADH-dehydrogenase module during assembly of the complex. Taken together, the findings provide new molecular-level understanding of the ndufs4-/- mouse model and complex I-linked mitochondrial disease.
Assuntos
Doença de Leigh , Doenças Mitocondriais , Animais , Camundongos , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Doença de Leigh/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , NAD/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismoRESUMO
Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure-activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.
Assuntos
Acetogeninas , Complexo I de Transporte de Elétrons , Acetogeninas/antagonistas & inibidores , Acetogeninas/metabolismo , Acetogeninas/farmacologia , Animais , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , NAD/metabolismo , Oxirredução , Relação Estrutura-Atividade , Ubiquinona/metabolismoRESUMO
The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their assignment as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC and F1Fo-ATP synthase complexes and sets the stage for future structural and drug discovery studies.
Assuntos
Transporte de Elétrons/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Toxoplasma/metabolismo , Animais , Humanos , Parasitos/metabolismo , Proteômica/métodos , Proteínas de Protozoários/metabolismo , Toxoplasmose/metabolismoRESUMO
Respiratory complex I is an essential metabolic enzyme that uses the energy from NADH oxidation and ubiquinone reduction to translocate protons across an energy transducing membrane and generate the proton motive force for ATP synthesis. Under specific conditions, complex I can also catalyze the reverse reaction, Δp-linked oxidation of ubiquinol to reduce NAD+ (or O2), known as reverse electron transfer (RET). Oxidative damage by reactive oxygen species generated during RET underpins ischemia reperfusion injury, but as RET relies on several converging metabolic pathways, little is known about its mechanism or regulation. Here, we demonstrate Δp-linked RET through complex I in a synthetic proteoliposome system for the first time, enabling complete kinetic characterization of RET catalysis. We further establish the capability of our system by showing how RET in the mammalian enzyme is regulated by the active-deactive transition and by evaluating RET by complex I from several species in which direct assessment has not been otherwise possible. We thus provide new insights into the reversibility of complex I catalysis, an important but little understood mechanistic and physiological feature.
Assuntos
Complexo I de Transporte de Elétrons , Elétrons , Animais , Catálise , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Mamíferos/metabolismo , NAD/metabolismo , OxirreduçãoRESUMO
Prostate cancer is one of the leading causes of cancer-related death in men. The identification of new therapeutics to selectively target prostate cancer cells is therefore vital. Recently, the rotenoids rotenone (1) and deguelin (2) were reported to selectively kill prostate cancer cells, and the inhibition of mitochondrial complex I was established as essential to their mechanism of action. However, these hydrophobic rotenoids readily cross the blood-brain barrier and induce symptoms characteristic of Parkinson's disease in animals. Since hydroxylated derivatives of 1 and 2 are more hydrophilic and less likely to readily cross the blood-brain barrier, 29 natural and unnatural hydroxylated derivatives of 1 and 2 were synthesized for evaluation. The inhibitory potency (IC50) of each derivative against complex I was measured, and its hydrophobicity (Slog10P) predicted. Amorphigenin (3), dalpanol (4), dihydroamorphigenin (5), and amorphigenol (6) were selected and evaluated in cell-based assays using C4-2 and C4-2B prostate cancer cells alongside control PNT2 prostate cells. These rotenoids inhibit complex I in cells, decrease oxygen consumption, and selectively inhibit the proliferation of prostate cancer cells, leaving control cells unaffected. The greatest selectivity and antiproliferative effects were observed with 3 and 5. The data highlight these molecules as promising therapeutic candidates for further evaluation in prostate cancer models.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Rotenona/análogos & derivados , Rotenona/farmacologia , Desacopladores/farmacologia , Animais , Barreira Hematoencefálica , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Humanos , Masculino , Membranas Mitocondriais/efeitos dos fármacos , Estrutura Molecular , Rotenona/química , Desacopladores/químicaRESUMO
Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ1) interactions in the yeast Ndi1-UQ1 complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.
Assuntos
Quinolonas/química , Quinona Redutases/antagonistas & inibidores , Quinona Redutases/química , Bactérias/enzimologia , Sítios de Ligação , Cristalografia , Desenho de Fármacos , Ligação de Hidrogênio , Ubiquinona/químicaRESUMO
Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.
Assuntos
Complexo I de Transporte de Elétrons/genética , Complexos de Proteínas Captadores de Luz/genética , Isoformas de Proteínas/genética , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação , Bovinos , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/economia , Complexo I de Transporte de Elétrons/isolamento & purificação , Humanos , Complexos de Proteínas Captadores de Luz/química , Mitocôndrias Cardíacas/química , Mitocôndrias Cardíacas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , RatosRESUMO
Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Transporte de Elétrons , Prótons , Animais , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/genética , Elétrons , Histidina/química , Histidina/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Oxirredução , Ubiquinona/metabolismoRESUMO
Dysfunctions in mitochondrial complex I (NADH:ubiquinone oxidoreductase) are both genetically and clinically highly diverse and a major cause of human mitochondrial diseases. The genetic determinants of individual clinical cases are increasingly being described, but how these genetic defects affect complex I on the molecular and cellular level, and have different clinical consequences in different individuals, is little understood. Furthermore, without molecular-level information innocent genetic variants may be misassigned as pathogenic. Here, we have used a yeast model system (Yarrowia lipolytica) to study the molecular consequences of 16 single amino acid substitutions, classified as pathogenic, in the NDUFV1 subunit of complex I. NDUFV1 binds the flavin cofactor that oxidizes NADH and is the site of complex I-mediated reactive oxygen species production. Seven mutations caused loss of complex I expression, suggesting they are detrimental but precluding further study. In two variants complex I was fully assembled but did not contain any flavin, and four mutations led to functionally compromised enzymes. Our study provides a molecular rationale for assignment of all these variants as pathogenic. However, three variants provided complex I that was functionally equivalent to the wild-type enzyme, challenging their assignment as pathogenic. By combining structural, bioinformatic and functional data, a simple scoring system for the initial evaluation of future NDUFV1 variants is proposed. Overall, our results broaden understanding of how mutations in this centrally important core subunit of complex I affect its function and provide a basis for understanding the role of NDUFV1 mutations in mitochondrial dysfunction.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Flavinas/metabolismo , NADH Desidrogenase/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , NADH Desidrogenase/química , NADH Desidrogenase/metabolismo , Ligação Proteica , Subunidades Proteicas , Yarrowia/genéticaRESUMO
BACKGROUND: The biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known. RESULTS: Here, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments. CONCLUSIONS: Biguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.
Assuntos
Adenilato Quinase/metabolismo , Biguanidas/química , Biguanidas/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fenformin/farmacologia , Ratos , Rotenona/farmacologia , SolubilidadeRESUMO
Complexâ I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone-10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10 analogues that partition into membranes and undergo side reactions. Herein, we present a self-assembled system without these limitations: proteoliposomes containing mammalian complexâ I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2 to Q10. AOX is present in excess, so complexâ I is completely rate determining and the Q10 pool is kept oxidized under steady-state catalysis. The system was used to measure a fully-defined K(M) value for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.
Assuntos
Complexo I de Transporte de Elétrons/química , Proteínas Mitocondriais/química , NAD/química , Oxirredutases/química , Proteínas de Plantas/química , Ubiquinona/química , Catálise , Transporte de Elétrons , OxirreduçãoRESUMO
The biguanide metformin is widely prescribed for Type II diabetes and has anti-neoplastic activity in laboratory models. Despite evidence that inhibition of mitochondrial respiratory complex I by metformin is the primary cause of its cell-lineage-specific actions and therapeutic effects, the molecular interaction(s) between metformin and complex I remain uncharacterized. In the present paper, we describe the effects of five pharmacologically relevant biguanides on oxidative phosphorylation in mammalian mitochondria. We report that biguanides inhibit complex I by inhibiting ubiquinone reduction (but not competitively) and, independently, stimulate reactive oxygen species production by the complex I flavin. Biguanides also inhibit mitochondrial ATP synthase, and two of them inhibit only ATP hydrolysis, not synthesis. Thus we identify biguanides as a new class of complex I and ATP synthase inhibitor. By comparing biguanide effects on isolated complex I and cultured cells, we distinguish three anti-diabetic and potentially anti-neoplastic biguanides (metformin, buformin and phenformin) from two anti-malarial biguanides (cycloguanil and proguanil): the former are accumulated into mammalian mitochondria and affect oxidative phosphorylation, whereas the latter are excluded so act only on the parasite. Our mechanistic and pharmacokinetic insights are relevant to understanding and developing the role of biguanides in new and existing therapeutic applications, including cancer, diabetes and malaria.
Assuntos
Biguanidas/farmacologia , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Metformina/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Biguanidas/uso terapêutico , Bovinos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Células Hep G2 , Humanos , Metformina/uso terapêutico , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Plasmodium/efeitos dos fármacos , Proguanil/farmacologia , Ratos , Traumatismo por Reperfusão/prevenção & controle , Triazinas/farmacologiaRESUMO
NADH:quinone oxidoreductase (complex I) couples NADH oxidation and quinone reduction to proton translocation across an energy-transducing membrane. All complexes I contain a flavin to oxidize NADH, seven iron-sulfur clusters to transfer electrons from the flavin to quinone and an eighth cluster (N1a) on the opposite side of the flavin. The role of cluster N1a is unknown, but Escherichia coli complex I has an unusually high-potential cluster N1a and its reduced flavin produces H2O2, not superoxide, suggesting that cluster N1a may affect reactive oxygen species production. In the present study, we combine protein film voltammetry with mutagenesis in overproduced N1a-binding subunits to identify two residues that switch N1a between its high- (E. coli, valine and asparagine) and low- (Bos taurus and Yarrowia lipolytica, proline and methionine) potential forms. The mutations were incorporated into E. coli complex I: cluster N1a could no longer be reduced by NADH, but H2O2 and superoxide production were unaffected. The reverse mutations (that increase the potential by ~0.16 V) were incorporated into Y. lipolytica complex I, but N1a was still not reduced by NADH. We conclude that cluster N1a does not affect reactive oxygen species production by the complex I flavin; it is probably required for enzyme assembly or stability.
Assuntos
Proteínas de Bactérias/química , Complexo I de Transporte de Elétrons/química , Animais , Proteínas de Bactérias/genética , Bovinos , Dinitrocresóis/química , Complexo I de Transporte de Elétrons/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Ligação de Hidrogênio , Cinética , Mutação , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/genética , Espécies Reativas de Oxigênio/química , Yarrowia/enzimologia , Yarrowia/genéticaRESUMO
The molecular mode of action of biguanides, including the drug metformin, which is widely used in the treatment of diabetes, is incompletely characterized. Here, we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo-electron microscopy and enzyme kinetics. We interpret these data to explain the selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel, and an additional binding site is in a pocket on the intermembrane-space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable the rational design of medicinal biguanides.
Assuntos
Biguanidas , Complexo I de Transporte de Elétrons , Animais , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Metformina/farmacologia , Mitocôndrias/metabolismo , Biguanidas/farmacologiaRESUMO
Respiratory complex I, a key enzyme in mammalian metabolism, captures the energy released by reduction of ubiquinone by NADH to drive protons across the inner mitochondrial membrane, generating the proton-motive force for ATP synthesis. Despite remarkable advances in structural knowledge of this complicated membrane-bound enzyme, its mechanism of catalysis remains controversial. In particular, how ubiquinone reduction is coupled to proton pumping and the pathways and mechanisms of proton translocation are contested. We present a 2.4-Å resolution cryo-EM structure of complex I from mouse heart mitochondria in the closed, active (ready-to-go) resting state, with 2945 water molecules modeled. By analyzing the networks of charged and polar residues and water molecules present, we evaluate candidate pathways for proton transfer through the enzyme, for the chemical protons for ubiquinone reduction, and for the protons transported across the membrane. Last, we compare our data to the predictions of extant mechanistic models, and identify key questions to answer in future work to test them.
Assuntos
Complexo I de Transporte de Elétrons , Prótons , Camundongos , Animais , Oxirredução , Complexo I de Transporte de Elétrons/metabolismo , Ubiquinona/química , Ubiquinona/metabolismo , Microscopia Crioeletrônica , Mamíferos/metabolismo , Água/metabolismoRESUMO
Current therapies for myeloproliferative neoplasms (MPNs) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem/progenitor cells (HSPCs). TAMARIN Phase-II, multicenter, single-arm clinical trial assessed tamoxifen's safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2V617F, CALRins5 or CALRdel52 peripheral blood allele burden ≥20% (EudraCT 2015-005497-38). 38 patients were recruited over 112w and 32 completed 24w-treatment. The study's A'herns success criteria were met as the primary outcome ( ≥ 50% reduction in mutant allele burden at 24w) was observed in 3/38 patients. Secondary outcomes included ≥25% reduction at 24w (5/38), ≥50% reduction at 12w (0/38), thrombotic events (2/38), toxicities, hematological response, proportion of patients in each IWG-MRT response category and ELN response criteria. As exploratory outcomes, baseline analysis of HSPC transcriptome segregates responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis shows high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which are downregulated by tamoxifen. We further demonstrate in preclinical studies that in JAK2V617F+ cells, 4-hydroxytamoxifen inhibits mitochondrial complex-I, activates integrated stress response and decreases pathogenic JAK2-signaling. These results warrant further investigation of tamoxifen in MPN, with careful consideration of thrombotic risk.
Assuntos
Transtornos Mieloproliferativos , Neoplasias , Humanos , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Neoplasias/metabolismo , Tamoxifeno/uso terapêutico , Tamoxifeno/metabolismo , Mutação , Calreticulina/genética , Calreticulina/metabolismoRESUMO
In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-cluster chain extending from the flavin) are (at least partially) reduced by NADH. The other three clusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyme: they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use Mössbauer spectroscopy on (57)Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent clusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential clusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential clusters. The implications for intramolecular electron transfer along the extended chain of cofactors in complex I are discussed.
Assuntos
Complexo I de Transporte de Elétrons/química , Proteínas Ferro-Enxofre/química , Quinona Redutases/química , Espectroscopia de Mossbauer , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Compostos Férricos/química , Compostos Ferrosos/química , Proteínas Fúngicas/química , Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/química , Oxirredução , Quinona Redutases/antagonistas & inibidores , Quinona Redutases/metabolismo , Espectroscopia de Mossbauer/métodos , Yarrowia/enzimologia , Yarrowia/metabolismoRESUMO
Respiratory complex I (NADH:quinone oxidoreductase) is an entry point to the electron transport chain in the mitochondria of many eukaryotes. It is a large, multisubunit enzyme with a hydrophilic domain in the matrix and a hydrophobic domain in the mitochondrial inner membrane. Here we present a comprehensive analysis of the protein composition and post-translational modifications of complex I from Pichia pastoris, using a combination of proteomic and bioinformatic approaches. Forty-one subunits were identified in P. pastoris complex I, comprising the 14 core (conserved) subunits and 27 supernumerary subunits; seven of the core subunits are mitochondrial encoded. Three of the supernumerary subunits (named NUSM, NUTM, and NUUM) have not been observed previously in any species of complex I. However, homologues to all three of them are present in either Yarrowia lipolytica or Pichia angusta complex I. P. pastoris complex I has 39 subunits in common with Y. lipolytica complex I, 37 in common with N. crassa complex I, and 35 in common with the bovine enzyme. The mitochondrial encoded subunits (translated by the mold mitochondrial genetic code) retain their N-α-formyl methionine residues. At least eight subunits are N-α-acetylated, but the N-terminal modifications of the nuclear encoded subunits are not well-conserved. A combination of two methods of protein separation (SDS-PAGE and HPLC) and three different mass spectrometry techniques (peptide mass fingerprinting, tandem MS and molecular mass measurements) were required to define the protein complement of P. pastoris complex I. This requirement highlights the need for inclusive and comprehensive strategies for the characterization of challenging membrane-bound protein complexes containing both hydrophilic and hydrophobic components.
Assuntos
Complexo I de Transporte de Elétrons/química , Pichia/genética , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Complexo I de Transporte de Elétrons/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, the mechanisms of Q10 reduction and energy transduction remain controversial. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the 'active' (ready-to-go) resting state together with a matching substrate-free structure, and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 binding pose. By comparing ligand-bound and ligand-free forms of the 'deactive' resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.
Assuntos
Complexo I de Transporte de Elétrons , Ubiquinona , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/metabolismo , Mamíferos/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Ubiquinona/metabolismoRESUMO
OBJECTIVES: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney condition, accounting for 7%-10% of patients with kidney failure. Fundamental basic science and clinical research on ADPKD is underway worldwide but no one has yet considered which areas should be prioritised to maximise returns from limited future funding. The Polycystic Kidney Disease Charity began a priority setting partnership with the James Lind Alliance (JLA) in the UK in 2019-2020 to identify areas of uncertainty in the ADPKD care pathway and allow patients, carers and healthcare professionals to rank the 10 most important questions for research. DESIGN: The scope covered ADPKD diagnosis and management, identifying new treatments to prevent/slow disease progression and practical, integrated patient support (https://pkdcharity.org.uk/research/for-researchers/adpkd-research-priorities). We used adapted JLA methodology. Initially, an independent information specialist collated uncertainties in ADPKD care from recent consensus conference proceedings and additional literature. These were refined into indicative questions with Steering Group oversight. Finally, the 10 most important questions were established via a survey and online consensus workshop. SETTING: UK. PARTICIPANTS: 747 survey respondents (76% patients, 13% carers, 11% healthcare professionals); 23 workshop attendees. RESULTS: 117 uncertainties in ADPKD care were identified and refined into 35 indicative questions. A shortlist of 17 questions was established through the survey. Workshop participants reached agreement on the top 10 ranking. The top three questions prioritised by patients, carers and healthcare professionals centred around slowing disease progression, identifying persons for early treatment and organising care to improve outcomes. CONCLUSIONS: Our shortlist reflects the varied physical, psychological and practical challenges of living with and treating ADPKD, and perceived gaps in knowledge that impair optimal care. We propose that future ADPKD research funding takes these priorities into account to focus on the most important areas and to maximise improvements in ADPKD outcomes.