Helicobacter pylori employs a general protein glycosylation system for the modification of outer membrane adhesins.

Helicobacter pylori infection is associated with the development of several gastric diseases including gastric cancer. To reach a long-term colonization in the host stomach, H. pylori employs multiple outer membrane adhesins for binding to the gastric mucosa. However, due to the redundancy of adhesins that complement the adhesive function of bacteria, targeting each individual adhesin alone usually achieves nonideal outcomes for preventing bacterial adhesion. Here, we report that key adhesins AlpA/B and BabA/B in H. pylori are modified by glycans and display a two-step molecular weight upshift pattern from the cytoplasm to the inner membrane and from the inner membrane to the outer membrane. Nevertheless, this upshift pattern is missing when the expression of some enzymes related to lipopolysaccharide (LPS) biosynthesis, including the LPS O-antigen assembly and ligation enzymes WecA, Wzk, and WaaL, is disrupted, indicating that the underlying mechanisms and the involved enzymes for the adhesin glycosylation are partially shared with the LPS biosynthesis. Loss of the adhesin glycosylation not only reduces the protease resistance and the stability of the tested adhesins but also changes the adhesin-binding ability. In addition, mutations in the LPS biosynthesis cause a significant reduction in bacterial adhesion in the in vitro cell-line model. The current findings reveal that H. pylori employs a general protein glycosylation system related to LPS biosynthesis for adhesin modification and its biological significance. The enzymes required for adhesin glycosylation rather than the adhesins themselves are potentially better drug targets for preventing or treating H. pylori infection.

The Effects of HP0044 and HP1275 Knockout Mutations on the Structure and Function of Lipopolysaccharide in Helicobacter pylori Strain 26695.

Helicobacter pylori infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Due to the prevalence and severeness of H. pylori infection, a thorough understanding of this pathogen is necessary. Lipopolysaccharide, one of the major virulence factors of H. pylori, can exert immunomodulating and immunostimulating functions on the host. In this study, the HP0044 and HP1275 genes were under investigation. These two genes potentially encode GDP-D-mannose dehydratase (GMD) and phosphomannomutase (PMM)/phosphoglucomutase (PGM), respectively, and are involved in the biosynthesis of fucose. HP0044 and HP1275 knockout mutants were generated; both mutants displayed a truncated LPS, suggesting that the encoded enzymes are not only involved in fucose production but are also important for LPS construction. In addition, these two gene knockout mutants exhibited retarded growth, increased surface hydrophobicity and autoaggregation as well as being more sensitive to the detergent SDS and the antibiotic novobiocin. Furthermore, the LPS-defective mutants also had significantly reduced bacterial infection, adhesion and internalization in the in vitro cell line model. Moreover, disruptions of the HP0044 and HP1275 genes in H. pylori altered protein sorting into outer membrane vesicles. The critical roles of HP0044 and HP1275 in LPS biosynthesis, bacterial fitness and pathogenesis make them attractive candidates for drug inventions against H. pylori infection.

LC3A-mediated autophagy regulates lung cancer cell plasticity.

ABBREVIATIONS: ATG14: autophagy related 14; CDH2: cadherin 2; ChIP-qPCR: chromatin immunoprecipitation quantitative polymerase chain reaction; CQ: chloroquine; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; EPCAM: epithelial cell adhesion molecule; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NDUFV2: NADH:ubiquinone oxidoreductase core subunit V2; OCR: oxygen consumption rate; ROS: reactive oxygen species; RT-qPCR: reverse-transcriptase quantitative polymerase chain reaction; SC: scrambled control; shRNA: short hairpin RNA; SNAI2: snail family transcriptional repressor 2; SOX2: SRY-box transcription factor 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-ß: transforming growth factor beta; TOMM20: translocase of outer mitochondrial membrane 20; ZEB1: zinc finger E-box binding homeobox 1.

TAT-Conjugated NDUFS8 Can Be Transduced into Mitochondria in a Membrane-Potential-Independent Manner and Rescue Complex I Deficiency.

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

Antimicrobial Activity of the Peptide LfcinB15 against Candida albicans.

Lactoferricin (Lfcin) is an amphipathic, cationic peptide derived from proteolytic cleavage of the N-lobe of lactoferrin (Lf). Lfcin and its derivatives possess broad-spectrum antibacterial and antifungal activities. However, unlike their antibacterial functions, the modes of action of Lfcin and its derivatives against pathogenic fungi are less well understood. In this study, the mechanisms of LfcinB15, a derivative of bovine Lfcin, against Candida albicans were, therefore, extensively investigated. LfcinB15 exhibited inhibitory activity against planktonic cells, biofilm cells, and clinical isolates of C. albicans and non-albicans Candida species. We further demonstrated that LfcinB15 is localized on the cell surface and vacuoles of C. albicans cells. Moreover, LfcinB15 uses several different methods to kill C. albicans, including disturbing the cell membrane, inducing reactive oxygen species (ROS) generation, and causing mitochondrial dysfunction. Finally, the Hog1 and Mkc1 mitogen-activated protein kinases were both activated in C. albicans cells in response to LfcinB15. These findings help us to obtain more insight into the complex mechanisms used by LfcinB15 and other Lfcin-derived peptides to fight fungal pathogens.

Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence.

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the ß-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA, VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells.

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.

Novel mitochondrial complex I-inhibiting peptides restrain NADH dehydrogenase activity.

The emergence of drug-resistant fungal pathogens is becoming increasingly serious due to overuse of antifungals. Antimicrobial peptides have potent activity against a broad spectrum of pathogens, including fungi, and are considered a potential new class of antifungals. In this study, we examined the activities of the newly designed peptides P-113Du and P-113Tri, together with their parental peptide P-113, against the human fungal pathogen Candida albicans. The results showed that these peptides inhibit mitochondrial complex I, specifically NADH dehydrogenase, of the electron transport chain. Moreover, P-113Du and P-113Tri also block alternative NADH dehydrogenases. Currently, most inhibitors of the mitochondrial complex I are small molecules or artificially-designed antibodies. Here, we demonstrated novel functions of antimicrobial peptides in inhibiting the mitochondrial complex I of C. albicans, providing insight in the development of new antifungal agents.

Helicobacter pylori induces intracellular galectin-8 aggregation around damaged lysosomes within gastric epithelial cells in a host O-glycan-dependent manner.

Galectin-8, a beta-galactoside-binding lectin, is upregulated in the gastric tissues of rhesus macaques infected with Helicobacter pylori. In this study, we found that H. pylori infection triggers intracellular galectin-8 aggregation in human-derived AGS gastric epithelial cells, and that these aggregates colocalize with lysosomes. Notably, this aggregation is markedly reduced following the attenuation of host O-glycan processing. This indicates that H. pylori infection induces lysosomal damage, which in turn results in the accumulation of cytosolic galectin-8 around damaged lysosomes through the recognition of exposed vacuolar host O-glycans. H. pylori-induced galectin-8 aggregates also colocalize with autophagosomes, and galectin-8 ablation reduces the activation of autophagy by H. pylori. This suggests that galectin-8 aggregates may enhance autophagy activity in infected cells. We also observed that both autophagy and NDP52, an autophagy adapter, contribute to the augmentation of galectin-8 aggregation by H. pylori. Additionally, vacuolating cytotoxin A, a secreted H. pylori cytotoxin, may contribute to the increased galectin-8 aggregation and elevated autophagy response in infected cells. Collectively, these results suggest that H. pylori promotes intracellular galectin-8 aggregation, and that galectin-8 aggregation and autophagy may reciprocally regulate each other during infection.

Stromal C-type lectin receptor COLEC12 integrates H. pylori, PGE2-EP2/4 axis and innate immunity in gastric diseases.

Tissue stroma is known to be important in regulating Hp-mediated inflammation, but its interaction with Hp and dendritic cells (DCs) remains to be determined. To this end, the potential crosstalk between H. pylori (Hp) infected gastric stromal cells (Hp-GSCs) and DCs was investigated. Primary GSCs from cancerous and adjacent normal tissues were generated from gastric cancer patients, and monocyte-derived DCs were obtained from healthy individuals. Levels of cytokines and prostaglandin E2 (PGE2) were measured by ELISA, and C-type lectin expression in GSCs was assessed by flow cytometry and immunohistochemistry. In a trans-well co-culture system, significantly upregulated DC-derived IL-23 expression was found when DCs were co-cultured with Hp-infected GSCs (Hp-GSCs). Further, PGE2 from Hp-GSCs was discovered to possess the priming effect, which could be inhibited by anti-COLEC12 (Collectin subfamily member 12) Abs, COLEC12 knockdown or when alpha3-fucosyltransferase-null (futB; HP0651) strain of Hp was used. Also, the expression of COLEC12 was co-localized with CD90+ stromal cells in cancerous tissues. Hp-GSCs-conditioned DCs were able to induce the expression of IL-17 from CD4+ T cells, which could be inhibited by IL-23-neutralizing Abs. These results suggested the importance of COLEC12 as a receptor involved in Hp-stromal cell interaction and its subsequent conditioning effect on DCs.

Functional characterization of Helicobacter pylori 26695 sedoheptulose 7-phosphate isomerase encoded by hp0857 and its association with lipopolysaccharide biosynthesis and adhesion.

Helicobacter pylori is a notorious human pathogen and the appearance of antibiotic resistance of this bacterium has posed a serious threat to human health. Lipopolysaccharide (LPS) is a key virulence factor and plays important roles in pathogenesis of H. pylori infection. Sedoheptulose 7-phosphate isomerase (GmhA), as an enzyme participating in the first step of heptose biosynthesis, is indispensable for the formation of inner core oligosaccharide of LPS. In this study, we cloned one putative gmhA ortholog, hp0857, from H. pylori 26695 and overexpressed it in Eschericha coli. Based on the results of molecular weight determination, the recombinant HP0857 is likely a homodimer. Analysis of enzymatic kinetic properties of this protein confirmed that hp0857 is indeed encoded a phosphoheptose isomerase which can utilize sedoheptulose 7-phosphate as the substrate in the ADP-L-glycero-D-manno-heptose (ADP- L,D-Hep) biosynthesis pathway. We also generated an HP0857 knockout mutant and explored its phenotypic changes. This mutant exhibited a decreased growth rate and displayed a "deep rough" type of LPS structure. In addition, it also had a slight decrease in its motility and was more susceptible to hydrophobic antibiotic novobiocin and detergents Triton X-100 and SDS. Furthermore, the adhesive capacity of the HP0857 knockout mutant to AGS cells was reduced significantly, and most of the infected cells didn't show a classic hummingbird phenotype. However, complementation of the HP0857 knockout mutation restored most of these phenotypic changes. In conclusion, we demonstrated that HP0857 protein is essential for inner core biosynthesis of H. pylori LPS and is a potential target for developing new antimicrobial agents against H. pylori infection.

Low-dose ionizing radiation induces mitochondrial fusion and increases expression of mitochondrial complexes I and III in hippocampal neurons.

High energy ionizing radiation can cause DNA damage and cell death. During clinical radiation therapy, the radiation dose could range from 15 to 60 Gy depending on targets. While 2 Gy radiation has been shown to cause cancer cell death, studies also suggest a protective potential by low dose radiation. In this study, we examined the effect of 0.2-2 Gy radiation on hippocampal neurons. Low dose 0.2 Gy radiation treatment increased the levels of MTT. Since hippocampal neurons are post-mitotic, this result reveals a possibility that 0.2 Gy irradiation may increase mitochondrial activity to cope with stimuli. Maintaining neural plasticity is an energy-demanding process that requires high efficient mitochondrial function. We thus hypothesized that low dose radiation may regulate mitochondrial dynamics and function to ensure survival of neurons. Our results showed that five days after 0.2 Gy irradiation, no obvious changes on neuronal survival, neuronal synapses, membrane potential of mitochondria, reactive oxygen species levels, and mitochondrial DNA copy numbers. Interestingly, 0.2 Gy irradiation promoted the mitochondria fusion, resulting in part from the increased level of a mitochondrial fusion protein, Mfn2, and inhibition of Drp1 fission protein trafficking to the mitochondria. Accompanying with the increased mitochondrial fusion, the expressions of complexes I and III of the electron transport chain were also increased. These findings suggest that, hippocampal neurons undergo increased mitochondrial fusion to modulate cellular activity as an adaptive mechanism in response to low dose radiation.

Therapeutic applications of the TAT-mediated protein transduction system for complex I deficiency and other mitochondrial diseases.

Among the five enzyme complexes in the oxidative phosphorylation system, NADH-coenzyme Q oxidoreductase (also called complex I) is the largest, most intricate, and least understood. This enzyme complex spans the inner mitochondrial membrane and catalyzes the first step of electron transfer by the oxidation of NADH, and thereby provides two electrons for the reduction of quinone to quinol. Complex I deficiency is associated with many severe mitochondrial diseases, including Leber hereditary optic neuropathy and Leigh syndrome. However, to date, conventional treatments for the majority of genetic mitochondrial diseases are only palliative. Developing a reliable and convenient therapeutic approach is therefore considered to be an urgent need. Targeted proteins fused with the protein transduction domain of human immunodeficiency virus 1 transactivator of transcription (TAT) have been shown to enter cells by crossing plasma membranes while retaining their biological activities. Recent developments show that, in fusion with mitochondrial targeting sequences (MTSs), TAT-MTS-bound cargo can be correctly transported into mitochondria and restore the missing function of the cargo protein in patients' cells. The available evidence suggests that the TAT-mediated protein transduction system holds great promise as a potential therapeutic approach to treat complex I deficiency, as well as other mitochondrial diseases.

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.

Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy.

BACKGROUND: NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy. METHODS: A series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease. RESULTS: We identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability. CONCLUSIONS: The mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase.

Helicobacter pylori is a bacterium that causes chronic active gastritis and peptic ulcers. Drugs targeting H. pylori phosphopantetheine adenylyltransferase (HpPPAT), which is involved in CoA biosynthesis, may be useful. Herein, we report the expression in Escherichia coli and purification of recombinant HpPPAT and describe a crystal structure for an HpPPAT/CoA complex. As is the case for E. coli PPAT (EcPPAT), HpPPAT is hexameric in solution and as a crystal. Each protomer has a well-packed dinucleotide-binding fold in which CoA binds. Structural characterisation demonstrated that CoA derived from the E. coli expression system bound tightly to HpPPAT, presumably to initiate feedback inhibition. However, the interactions between the active-site residues of HpPPAT and CoA are not identical to those of other PPATs. Finally, CoA binding affects HpPPAT thermal denaturation.

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells.

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-L-glycero-D-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP+, instead of NAD+, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.

The membrane subunit NuoL(ND5) is involved in the indirect proton pumping mechanism of Escherichia coli complex I.

Complex I pumps protons across the membrane by using downhill redox energy. Here, to investigate the proton pumping mechanism by complex I, we focused on the largest transmembrane subunit NuoL (Escherichia coli ND5 homolog). NuoL/ND5 is believed to have H(+) translocation site(s), because of a high sequence similarity to multi-subunit Na(+)/H(+) antiporters. We mutated thirteen highly conserved residues between NuoL/ND5 and MrpA of Na(+)/H(+) antiporters in the chromosomal nuoL gene. The dNADH oxidase activities in mutant membranes were mostly at the control level or modestly reduced, except mutants of Glu-144, Lys-229, and Lys-399. In contrast, the peripheral dNADH-K(3)Fe(CN)(6) reductase activities basically remained unchanged in all the NuoL mutants, suggesting that the peripheral arm of complex I was not affected by point mutations in NuoL. The proton pumping efficiency (the ratio of H(+)/e(-)), however, was decreased in most NuoL mutants by 30-50%, while the IC(50) values for asimicin (a potent complex I inhibitor) remained unchanged. This suggests that the H(+)/e(-) stoichiometry has changed from 4H(+)/2e(-) to 3H(+) or 2H(+)/2e(-) without affecting the direct coupling site. Furthermore, 50 µm of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific inhibitor for Na(+)/H(+) antiporters, caused a 38 ± 5% decrease in the initial H(+) pump activity in the wild type, while no change was observed in D178N, D303A, and D400A mutants where the H(+) pumping efficiency had already been significantly decreased. The electron transfer activities were basically unaffected by EIPA in both control and mutants. Taken together, our data strongly indicate that the NuoL subunit is involved in the indirect coupling mechanism.

Can a single subunit yeast NADH dehydrogenase (Ndi1) remedy diseases caused by respiratory complex I defects?

The proton-translocating NADH-quinone oxidoreductase (complex I) is one of five enzyme complexes in the oxidative phosphorylation system in mammalian mitochondria. Complex I is composed of 46 different subunits, 7 of which are encoded by mitochondrial DNA. Defects of complex I are involved in many human mitochondrial diseases; therefore, the authors proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into 10 mammalian cell lines (two of which were complex I-deficient mutants). The expressed Ndi1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional, and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species. It was further shown that the Ndi1 protein can be functionally expressed in tissues such as skeletal muscles and brain of rodents. The Ndi1 expression scarcely induced an inflammatory response as assessed by hematoxylin and eosin (H&E) staining. The Ndi1 protein expressed in the substantia nigra (SN) elicited protective effects against neurodegeneration caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The Ndi1 protein has a great potential as a molecular remedy for complex I deficiencies.

Characterization of the membrane domain subunit NuoK (ND4L) of the NADH-quinone oxidoreductase from Escherichia coli.

The ND4L subunit is the smallest mitochondrial DNA-encoded subunit of the proton-translocating NADH-quinone oxidoreductase (complex I). In an attempt to study the functional and structural roles of the NuoK subunit (the Escherichia coli homologue of ND4L) of the bacterial NADH-quinone oxidoreductase (NDH-1), we have performed a series of site-specific mutations on the nuoK gene of the NDH-1 operon by using the homologous recombination technique. The amino acid residues we targeted included two highly conserved glutamic acids that are presumably located in the middle of the membrane and several arginine residues that are predicted to be on the cytosolic side. All point mutants examined had fully assembled NDH-1 as detected by blue-native gel electrophoresis and immunostaining. Mutations of nearly perfectly conserved Glu-36 lead to almost null activities of coupled electron transfer with a concomitant loss of generation of electrochemical gradient. A significant diminution of the coupled activities was also observed with mutations of another highly conserved residue, Glu-72. These results may suggest that both membrane-embedded acidic residues are important for the coupling mechanism of NDH-1. Furthermore, a severe impairment of the coupled activities occurred when two vicinal arginine residues on a cytosolic loop were simultaneously mutated. Possible roles of these arginine residues and other conserved residues in the NuoK subunit for NDH-1 function were discussed.