Structural insights into respiratory complex I deficiency and assembly from the mitochondrial disease-related ndufs4-/- mouse.

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.

Truncal Plane Blocks for Implantation of Cardiac Resynchronization Devices: A Systematic Review.

Patients undergoing subcutaneous implantable cardioverter-defibrillator (S-ICD) placement can experience significant perioperative pain. General anesthesia is traditionally used for S-ICD placement and is associated with increased risk. Truncal plane blocks (TPBs) and sedation offer an alternative for adequate analgesia while avoiding hemodynamic compromise related to general anesthesia. A comprehensive evidence search utilized PubMed, CINAHL, Google Scholar, EBSCOhost, US National Library of Medicine Clinical Trials, and Medline Complete databases and the evidence examined the efficacy of TPBs in S-ICD placement. The quality of evidence was assessed using the guidelines described in the Johns Hopkins Nursing Evidence-Based Practice Model. Three randomized-controlled trials, four nonrandomized experimental studies, two nonexperimental studies, and three case studies totaling 379 patients were reviewed. Ultrasound-guided TPBs with sedation demonstrated superior analgesic efficacy for S-ICD procedures. Hemodynamics marginally deviated from baseline values and were well tolerated by patients. The evidence suggests that TPBs provide adequate analgesia during intraoperative and postoperative periods. TPBs are effective in reducing pain scores and opioid consumption postoperatively. Although there were no significant changes in hemodynamic values, more research should be conducted to evaluate the effects on intraoperative hemodynamics.

Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis.

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.

Investigation of hydrated channels and proton pathways in a high-resolution cryo-EM structure of mammalian complex I.

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.

Structural basis of mammalian respiratory complex I inhibition by medicinal biguanides.

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.

Research priorities for autosomal dominant polycystic kidney disease: a UK priority setting partnership.

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.

Cryo-EM structures define ubiquinone-10 binding to mitochondrial complex I and conformational transitions accompanying Q-site occupancy.

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.

Reverse Electron Transfer by Respiratory Complex I Catalyzed in a Modular Proteoliposome System.

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.

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I.

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.

Complexome profile of Toxoplasma gondii mitochondria identifies divergent subunits of respiratory chain complexes including new subunits of cytochrome bc1 complex.

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.

Structural basis for a complex I mutation that blocks pathological ROS production.

Mitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia-reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the "deactive" state, usually formed only after prolonged inactivity. Despite its tendency to adopt the "deactive" state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

Structure of inhibitor-bound mammalian complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

Repression of LKB1 by miR-17∼92 Sensitizes MYC-Dependent Lymphoma to Biguanide Treatment.

Cancer cells display metabolic plasticity to survive stresses in the tumor microenvironment. Cellular adaptation to energetic stress is coordinated in part by signaling through the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Here, we demonstrate that miRNA-mediated silencing of LKB1 confers sensitivity of lymphoma cells to mitochondrial inhibition by biguanides. Using both classic (phenformin) and newly developed (IM156) biguanides, we demonstrate that elevated miR-17∼92 expression in Myc+ lymphoma cells promotes increased apoptosis to biguanide treatment in vitro and in vivo. This effect is driven by the miR-17-dependent silencing of LKB1, which reduces AMPK activation in response to complex I inhibition. Mechanistically, biguanide treatment induces metabolic stress in Myc+ lymphoma cells by inhibiting TCA cycle metabolism and mitochondrial respiration, exposing metabolic vulnerability. Finally, we demonstrate a direct correlation between miR-17∼92 expression and biguanide sensitivity in human cancer cells. Our results identify miR-17∼92 expression as a potential biomarker for biguanide sensitivity in malignancies.

Hydroxylated Rotenoids Selectively Inhibit the Proliferation of Prostate Cancer Cells.

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.

Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I.

Structure of the NDH-2 - HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors.

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.

Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction.

Using Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.

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.

The mechanism of catalysis by type-II NADH:quinone oxidoreductases.

Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundation for the development of therapeutic strategies.

Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

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.