Differential Effects of Yeast NADH Dehydrogenase (Ndi1) Expression on Mitochondrial Function and Inclusion Formation in a Cell Culture Model of Sporadic Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient's brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.

Application of Single Prolonged Stress Induces Post-traumatic Stress Disorder-like Characteristics in Mice.

We tried to clarify the applicability of a single prolonged stress (SPS) protocol as post-traumatic stress disorder (PTSD) model in mice. To investigate PTSD pathophysiology, we conducted hypothalamo-pituitary-adrenal (HPA) negative feedback testing at 1, 4, 8 and 12 weeks after the SPS by administrating a dexamethasone (DEX) suppression test. The SPS induced over-suppression of the HPA system by DEX treatment at 8 and 12 weeks. To investigate PTSD-like behavioral characteristics, we subjected mice to testing in a light/dark box (to assess anxiety), a Y-maze (working memory), a cliff avoidance (visual cognition), and an open field (locomotor activity) at 1, 4, 8 and 12 weeks after the SPS. In the light/dark box test, the SPS-applied mice spent significantly less time in the light box at 8 or 12 weeks. In the cliff avoidance test, the SPS-applied mice spent significantly less time in the open area at 1 week. However, in both the Y-maze test and the open field test, SPS-applied mice tended toward slight decreases in a time-dependent manner until 12 weeks. Therefore, SPS-applied mice may thus be useful for assessing characteristics relevant to PTSD that coincide with changes in the HPA axis.

Ubiquinone binding site of yeast NADH dehydrogenase revealed by structures binding novel competitive- and mixed-type inhibitors.

Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.

Conserved amino acid residues of the NuoD segment important for structure and function of Escherichia coli NDH-1 (complex I).

The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217-Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed.

Long-term evaluation of Leber's hereditary optic neuropathy-like symptoms in rotenone administered rats.

Leber's hereditary optic neuropathy (LHON) is an inherited disorder affecting the retinal ganglion cells (RGCs) and their axons that lead to the loss of central vision. This study is aimed at evaluating the LHON symptoms in rats administered with rotenone microspheres into the superior colliculus (SC). Optical coherence tomography (OCT) analysis showed substantial loss of retinal nerve fiber layer (RNFL) thickness in rotenone injected rats. Optokinetic testing in rotenone treated rats showed decrease in head-tracking response. Electrophysiological mapping of the SC surface demonstrated attenuation of visually evoked responses; however, no changes were observed in the ERG data. The progressive pattern of disease manifestation in rotenone administered rats demonstrated several similarities with human disease symptoms. These rats with LHON-like symptoms can serve as a model for future investigators to design and implement reliable tests to assess the beneficial effects of therapeutic interventions for LHON disease.

Essential regions in the membrane domain of bacterial complex I (NDH-1): the machinery for proton translocation.

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is the first and largest enzyme of the respiratory chain which has a central role in cellular energy production and is implicated in many human neurodegenerative diseases and aging. It is believed that the peripheral domain of complex I/NDH-1 transfers the electron from NADH to Quinone (Q) and the redox energy couples the proton translocation in the membrane domain. To investigate the mechanism of the proton translocation, in a series of works we have systematically studied all membrane subunits in the Escherichia coli NDH-1 by site-directed mutagenesis. In this mini-review, we have summarized our strategy and results of the mutagenesis by depicting residues essential for proton translocation, along with those for subunit connection. It is suggested that clues to understanding the driving forces of proton translocation lie in the similarities and differences of the membrane subunits, highlighting the communication of essential charged residues among the subunits. A possible proton translocation mechanism with all membrane subunits operating in unison is described.

Randomly selected suppressor mutations in genes for NADH : quinone oxidoreductase-1, which rescue motility of a Salmonella ubiquinone-biosynthesis mutant strain.

The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localized to genes for subunits of NADH : quinone oxidoreductase-1. Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of NADH : quinone oxidoreductase-1 and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain. Respiration was rescued and the suppressed mutant strains grew better in Luria-Bertani broth medium and could use l-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterized by reversed-phase HPLC. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased NADH : quinone oxidoreductase-1 levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Under certain growth conditions the suppressor mutations improved electron flow activity of NADH : quinone oxidoreductase-1 for cells bearing a ubiA deletion mutation.

Complex I inhibition in the visual pathway induces disorganization of the node of Ranvier.

Mitochondrial defects can have significant consequences on many aspects of neuronal physiology. In particular, deficiencies in the first enzyme complex of the mitochondrial respiratory chain (complex I) are considered to be involved in a number of human neurodegenerative diseases. The current work highlights a tight correlation between the inhibition of complex I and the state of axonal myelination of the optic nerve. Exposing the visual pathway of rats to rotenone, a complex I inhibitor, resulted in disorganization of the node of Ranvier. The structure and function of the node depend on specific cell adhesion molecules, among others, CASPR (contactin associated protein) and contactin. CASPR and contactin are both on the axonal surfaces and need to be associated to be able to anchor their myelin counterpart. Here we show that inhibition of mitochondrial complex I by rotenone in rats induces reactive oxygen species, disrupts the interaction of CASPR and contactin couple, and thus damages the organization and function of the node of Ranvier. Demyelination of the optic nerve occurs as a consequence which is accompanied by a loss of vision. The physiological impairment could be reversed by introducing an alternative NADH dehydrogenase to the mitochondria of the visual system. The restoration of the nodal structure was specifically correlated with visual recovery in the treated animal.

Energy transducing roles of antiporter-like subunits in Escherichia coli NDH-1 with main focus on subunit NuoN (ND2).

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (NLys(395)) is conserved in NuoL (LLys(399)) but is replaced by glutamic acid (MGlu(407)) in NuoM. Our mutation study on NLys(395) suggests that this residue participates in the proton translocation. Furthermore, we found that MGlu(407) is also essential and most likely interacts with conserved LArg(175). Glutamic acids, NGlu(133), MGlu(144), and LGlu(144), are corresponding residues. Unlike mutants of MGlu(144) and LGlu(144), mutation of NGlu(133) scarcely affected the energy-transducing activities. However, a double mutant of NGlu(133) and nearby KGlu(72) showed significant inhibition of these activities. This suggests that NGlu(133) bears a functional role similar to LGlu(144) and MGlu(144) but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that NLys(158) is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of NTM14 interacts with the Mß sheet located on the opposite side of helix HL. Taken together, the mechanism of H(+) translocation in NDH-1 is discussed.

Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression.

Despite advances in clinical therapy, metastasis remains the leading cause of death in breast cancer patients. Mutations in mitochondrial DNA, including those affecting complex I and oxidative phosphorylation, are found in breast tumors and could facilitate metastasis. This study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Specific enhancement of mitochondrial complex I activity inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, nonlethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression rendered tumor cells more aggressive and increased metastasis. The results translate into a new therapeutic strategy: enhancement of the NAD+/NADH balance through treatment with NAD+ precursors inhibited metastasis in xenograft models, increased animal survival, and strongly interfered with oncogene-driven breast cancer progression in the MMTV-PyMT mouse model. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression.

A phenotypic model recapitulating the neuropathology of Parkinson's disease.

This study was undertaken to develop a phenotypic model recapitulating the neuropathology of Parkinson's disease (PD). Such a model would show loss of dopamine in the basal ganglia, appearance of Lewy bodies, and the early stages of motor dysfunction. The model was developed by subcutaneously injecting biodegradable microspheres of rotenone, a complex I inhibitor in 8-9 month old, ovariectomized Long-Evans rats. Animals were observed for changes in body weight and motor activity. At the end of 11-12 weeks animals were euthanized and the brains examined for histopathological changes. Rotenone treated animals gain weight and appear normal and healthy as compared to controls but showed modest hypokinesia around 5-6 weeks posttreatment. Animals showed loss of dopaminergic (DA) neurons and the appearance of putative Lewy bodies in the substantia nigra. Neuroinflammation and oxidative stress were evidenced by the appearance of activated microglia, iron precipitates, and 8-oxo-2'-deoxyguanosine a major product of DNA oxidation. The dorsal striatum, the projection site of midbrain DA neurons, showed a significant reduction in tyrosine hydroxylase immunostaining, together with an increase in reactive astrocytes, an early sign of DA nerve terminal damage. Levels of vesicular monoamine transporter 2 (VMAT2) were significantly reduced in the dorsal striatum; however, there was an unexpected increase in dopamine transporter (DAT) levels. Old, ovariectomized females treated with rotenone microspheres present with normal weight gain and good health but a modest hypokinesia. Accompanying this behavioral phenotype are a constellation of neuropathologies characteristic of PD that include loss of DA neurons, microglia activation, oxidative damage to nuclear DNA, iron deposition, and appearance of putative Lewy bodies. This phenotypic model recapitulating the neuropathology of Parkinson's disease could provide insight into early mechanisms of pathogenesis and could aid in the identification of biomarkers to identify patients in early stage, PD.

Roles of subunit NuoK (ND4L) in the energy-transducing mechanism of Escherichia coli NDH-1 (NADH:quinone oxidoreductase).

The bacterial H(+)-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1-3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue ((K)Glu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue ((K)Glu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted (K)Glu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of (K)Glu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK ((K)Arg-25, (K)Arg-26, and (K)Asn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates.

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD(+)- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD(+) and Ndi1-UQ2 complexes show overlapping binding sites for the NAD(+) and quinone substrates.

Electron transfer in subunit NuoI (TYKY) of Escherichia coli NADH:quinone oxidoreductase (NDH-1).

Bacterial proton-translocating NADH:quinone oxidoreductase (NDH-1) consists of a peripheral and a membrane domain. The peripheral domain catalyzes the electron transfer from NADH to quinone through a chain of seven iron-sulfur (Fe/S) clusters. Subunit NuoI in the peripheral domain contains two [4Fe-4S] clusters (N6a and N6b) and plays a role in bridging the electron transfer from cluster N5 to the terminal cluster N2. We constructed mutants for eight individual Cys-coordinating Fe/S clusters. With the exception of C63S, all mutants had damaged architecture of NDH-1, suggesting that Cys-coordinating Fe/S clusters help maintain the NDH-1 structure. Studies of three mutants (C63S-coordinating N6a, P110A located near N6a, and P71A in the vicinity of N6b) were carried out using EPR measurement. These three mutations did not affect the EPR signals from [2Fe-2S] clusters and retained electron transfer activities. Signals at g(z) = 2.09 disappeared in C63S and P110A but not in P71A. Considering our data together with the available information, g(z,x) = 2.09, 1.88 signals are assigned to cluster N6a. It is of interest that, in terms of g(z,x) values, cluster N6a is similar to cluster N4. In addition, we investigated the residues (Ile-94 and Ile-100) that are predicted to serve as electron wires between N6a and N6b and between N6b and N2, respectively. Replacement of Ile-100 and Ile-94 with Ala/Gly did not affect the electron transfer activity significantly. It is concluded that conserved Ile-100 and Ile-94 are not essential for the electron transfer.

No immune responses by the expression of the yeast Ndi1 protein in rats.

BACKGROUND: The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host's immune system. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol. CONCLUSIONS/SIGNIFICANCE: We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.

Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli.

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.

Reaction mechanism of single subunit NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae: evidence for a ternary complex mechanism.

The flavoprotein rotenone-insensitive internal NADH-ubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin.

Protective Role of rAAV-NDI1, Serotype 5, in an Acute MPTP Mouse Parkinson's Model.

Defects in mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) have been implicated in a number of acquired and hereditary diseases including Leigh's syndrome and more recently Parkinson's disease. A limited number of strategies have been attempted to repair the damaged complex I with little or no success. We have recently shown that the non-proton-pumping, internal NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae (baker's yeast) can be successfully inserted into the mitochondria of mice and rats, and the enzyme was found to be fully active. Using recombinant adenoassociated virus vectors (serotype 5) carrying our NDI1 gene, we were able to express the Ndi1 protein in the substantia nigra (SN) of C57BL/6 mice with an expression period of two months. The results show that the AAV serotype 5 was highly efficient in expressing Ndi1 in the SN, when compared to a previous model using serotype 2, which led to nearly 100% protection when using an acute MPTP model. It is conceivable that the AAV-serotype5 carrying the NDI1 gene is a powerful tool for proof-of-concept study to demonstrate complex I defects as the causable factor in diseases of the brain.

Pivotal roles of three conserved carboxyl residues of the NuoC (30k) segment in the structural integrity of proton-translocating NADH-quinone oxidoreductase from Escherichia coli.

The prokaryotic proton-translocating NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzyme that contains 14 subunits (NuoA-NuoN or Nqo1-Nqo14). All subunits have their counterparts in the eukaryotic enzyme (complex I). NDH-1 consists of two domains: the peripheral arm (NuoB, -C, -D, -E, -F, -G, and -I) and the membrane arm (NuoA, -H, -J, -K, -L, -M, and -N). In Escherichia coli NDH-1, the hydrophilic subunits NuoC/Nqo5/30k and NuoD/Nqo4/49k are fused together in a single polypeptide as the NuoCD subunit. The NuoCD subunit is the only subunit that does not bear a cofactor in the peripheral arm. While some roles for inhibitor and quinone association have been reported for the NuoD segment, structural and functional roles of the NuoC segment remain mostly elusive. In this work, 14 highly conserved residues of the NuoC segment were mutated and 21 mutants were constructed using the chromosomal gene manipulation technique. From the enzymatic assays and immunochemical and blue-native gel analyses, it was found that residues Glu-138, Glu-140, and Asp-143 that are thought to be in the third α-helix are absolutely required for the energy-transducing NDH-1 activities and the assembly of the whole enzyme. Together with available information for the hydrophobic subunits, we propose that Glu-138, Glu-140, and Asp-143 of the NuoC segment may have a pivotal role in the structural stability of NDH-1.