Oxidoreductase Complex Research Articles

IACS-010759, a potent inhibitor of glycolysis-deficient hypoxic tumor cells, inhibits mitochondrial respiratory complex I through a unique mechanism

The small molecule IACS-010759 has been reported to potently inhibit the proliferation of glycolysis-deficient hypoxic tumor cells by interfering with the functions of mitochondrial NADH-ubiquinone oxidoreductase (complex I) without exhibiting cytotoxicity at tolerated doses in normal cells. Considering the significant cytotoxicity of conventional quinone-site inhibitors of complex I, such as piericidin and acetogenin families, we hypothesized that the mechanism of action of IACS-010759 on complex I differs from that of other known quinone-site inhibitors. To test this possibility, here we investigated IACS-010759's mechanism in bovine heart submitochondrial particles. We found that IACS-010759, like known quinone-site inhibitors, suppresses chemical modification by the tosyl reagent AL1 of Asp160 in the 49-kDa subunit, located deep in the interior of a previously proposed quinone-access channel. However, contrary to the other inhibitors, IACS-010759 direction-dependently inhibited forward and reverse electron transfer and did not suppress binding of the quinazoline-type inhibitor [125I]AzQ to the N terminus of the 49-kDa subunit. Photoaffinity labeling experiments revealed that the photoreactive derivative [125I]IACS-010759-PD1 binds to the middle of the membrane subunit ND1 and that inhibitors that bind to the 49-kDa or PSST subunit cannot suppress the binding. We conclude that IACS-010759's binding location in complex I differs from that of any other known inhibitor of the enzyme. Our findings, along with those from previous study, reveal that the mechanisms of action of complex I inhibitors with widely different chemical properties are more diverse than can be accounted for by the quinone-access channel model proposed by structural biology studies.

Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I

NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky "block" that is ∼13 Å across. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable with ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.

Generation and isolation of recombinant retinoid oxidoreductase complex.

All-trans-retinoic acid (RA) is a bioactive lipid that influences many processes in embryonic and adult tissues. Given its bioactive nature, cellular concentrations of this molecule are highly regulated. The oxidation of all-trans-retinol to all-trans-retinaldehyde represents the first and rate-limiting step of the RA synthesis pathway. As such, it is the target of mechanisms that fine-tune RA levels within the cell. RDH10 is one enzyme responsible for the oxidation of all-trans-retinol to all-trans-retinaldehyde, and together with the all-trans-retinaldehyde reductase DHRS3 forms an oligomeric protein complex. The resulting retinoid oxidoreductase complex (ROC) is bifunctional and has the capacity to regulate steady-state levels of the direct precursor of RA, all-trans-retinaldehyde. As ROC represents a major regulatory element within the RA synthesis pathway, it is essential that methods are in place that allow for the study of this complex. Here we describe the production and isolation of recombinant ROC using a baculovirus expression system. Recombinant proteins retain enzymatic activities in intact microsomes and can be affinity purified for analysis. These methods can be used to assist in the assessment of ROC properties and the regulation of this protein complex's functional attributes.

AGING AND MITOCHONDRIAL DISEASE: SHARED MECHANISMS AND THERAPIES?

Mitochondrial disease describes multiple pathologies characterized by a wide array of disease symptoms and severity, caused by mitochondrial dysfunction in one or multiple organs. Aging organisms display a similar variety of disease phenotypes, which are often characterized by mitochondrial impairment. Despite the heterogeneity of aging phenotypes, several interventions have been identified which can increase lifespan and delay the onset of age-related diseases in multiple organisms. Two age-delaying interventions, rapamycin and acarbose, dramatically suppress pathology in a mouse model of mitochondrial disease caused by depletion of the NADH-Ubiquinone Oxidoreductase Complex (Ndufs4-/-). This model recapitulates human Leigh syndrome, a childhood mitochondrial disease. Upon treatment with either drug, disease suppression is accompanied by a remodeling of nutrient metabolism and restoration of the NAD+/NADH ratio in the brain without affecting the electron transport chain. Thus, we propose that metabolic derangements induced by mitochondrial dysfunction may be a shared mechanism of aging and mitochondrial disease.

Bioinformatics identification of key candidate genes and pathways associated with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by autoantibody production and multi-system involvement, but the etiology is largely unclear. This study aimed to elucidate candidate genes and pathways involved in SLE. Three original datasets GSE72509, GSE20864, and GSE39088 were downloaded from Gene Expression Omnibus (GEO) and the data were further integrated and analyzed. Subsequently, differentially expressed genes (DEGs) between SLE patients and healthy people were identified. And then we performed gene ontology (GO) function and pathway enrichment analyses of common DEGs, and constructed a protein-protein interaction (PPI) network with STRING database. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was carried out to validate the expression levels of candidate genes in blood samples from SLE patients and healthy controls. In total, 321 common DEGs were identified in SLE patients compared with healthy controls, including 231 upregulated and 90 downregulated genes. GO function analysis revealed that 321 common DEGs were mainly enriched in innate immune response, defense response, cytokine-mediated signaling pathway, response to interferon-alpha, and I-kappaB kinase/NF-kappaB signaling. Additionally, pathway enrichment analysis indicated that DEGs were mainly enriched in several signaling pathways associated with immune system and apoptosis, including RIG-I-like receptor signaling pathway, antigen processing and presentation, and p53 signaling pathway. The expression levels of candidate genes RPL26L1, FBXW11, FOXO1, and SMAD7 were validated by RT-qPCR analysis. The four hub genes including RPL26L1, FBXW11, FOXO1, and SMAD7 may play key roles in the pathogenesis and development of SLE. RIG-I-like receptor signaling pathway, antigen processing and presentation pathway, and p53 signaling pathway may be closely implicated in SLE pathogenesis. Collectively, these results may provide valuable novel markers or targets for the diagnosis and treatment of SLE.Key Points• Integrated bioinformatics analysis of three profile datasets based on SLE patients and healthy controls was performed and 321 common DEGs were identified.• The 321 common DEGs were mainly enriched in biological processes related to immune responses and inflammatory responses, including innate immune response, defense response, cytokine-mediated signaling pathway, response to interferon-alpha, I-kappaB kinase/NF-kappaB signaling, whereas the three most significant cellular components were oxidoreductase complex, AIM2 inflammasome complex, and ubiquitin ligase complex.• KEGG pathway enrichment analysis indicated that common DEGs were mainly enriched in several signaling pathways associated with immune system and apoptosis, including RIG-I-like receptor signaling pathway, antigen processing and presentation, and p53 signaling pathway.• Candidate genes RPL26L1, FBXW11, FOXO1, and SMAD7 may be closely involved in the pathogenesis and development of SLE and may provide valuable novel markers or targets for the diagnosis and treatment of SLE.

Structural analysis of Borreliaburgdorferi periplasmic lipoprotein BB0365 involved in Lyme disease infection.

The periplasmic lipoprotein BB0365 of the Lyme disease agent Borreliaburgdorferi is expressed throughout mammalian infection and is essential for all phases of Lyme disease infection; its function, however, remains unknown. In the current study, our structural analysis of BB0365 revealed the same structural fold as that found in the NqrC and RnfG subunits of the NADH:quinone and ferredoxin:NAD+ sodium-translocating oxidoreductase complexes, which points to a potential role for BB0365 as a component of the sodium pump. Additionally, BB0365 coordinated Zn2+ by the His51, His55, His140 residues, and the Zn2+ -binding site indicates that BB0365 could act as a potential metalloenzyme; therefore, this structure narrows down the potential functions of BB0365, an essential protein for B.burgdorferi to cause Lyme disease.

Engineered Thermoanaerobacterium aotearoense with nfnAB knockout for improved hydrogen\xa0production from lignocellulose hydrolysates

BackgroundAs a renewable and clean energy carrier, the production of biohydrogen from low-value feedstock such as lignocellulose has increasingly garnered interest. The NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB) complex catalyzes electron transfer between reduced ferredoxin and NAD(P)+, which is critical for production of NAD(P)H-dependent products such as hydrogen and ethanol. In this study, the effects on end-product formation of deletion of nfnAB from Thermoanaerobacterium aotearoense SCUT27 were investigated.ResultsCompared with the parental strain, the NADH/NAD+ ratio in the ∆nfnAB mutant was increased. The concentration of hydrogen and ethanol produced increased by (41.1 ± 2.37)% (p < 0.01) and (13.24 ± 1.12)% (p < 0.01), respectively, while the lactic acid concentration decreased by (11.88 ± 0.96)% (p < 0.01) when the ∆nfnAB mutant used glucose as sole carbon source. No obvious inhibition effect was observed for either SCUT27 or SCUT27/∆nfnAB when six types of lignocellulose hydrolysate pretreated with dilute acid were used for hydrogen production. Notably, the SCUT27/∆nfnAB mutant produced 190.63–209.31 mmol/L hydrogen, with a yield of 1.66–1.77 mol/mol and productivity of 12.71–13.95 mmol/L h from nonsterilized rice straw and corn cob hydrolysates pretreated with dilute acid.ConclusionsThe T. aotearoense SCUT27/∆nfnAB mutant showed higher hydrogen yield and productivity compared with those of the parental strain. Hence, we demonstrate that deletion of nfnAB from T. aotearoense SCUT27 is an effective approach to improve hydrogen production by redirecting the electron flux, and SCUT27/∆nfnAB is a promising candidate strain for efficient biohydrogen production from lignocellulosic hydrolysates.

A proteomic-based approach for the search of biomarkers in Iberian wild deer (Cervus elaphus) as indicators of meat quality

The positive perception of deer meat among consumers is growing. Consequently, further efforts are needed to control its meat quality. Specifically, the aim of this study was to search protein biomarkers in connection with tenderness and intramuscular fat (IMF) content of Iberian wild red deer (Cervus elaphus) meat. Twelve venison samples classified as tender, intermediate and tough meat, as well as with different IMF were studied, using quantitative proteomic by SWATH-MS combined with bioinformatic analyses. The study of muscle proteome showed significant differences among these three groups of meat samples, since five differentially abundant proteins (IVD, LAMB1, MYL3, SDHC and SDHA) and six (FABP4, IVD, LAMB1, MYL3, CRYZ and SERPINB6) were strongly correlated with tenderness and IMF, respectively. The proteomic approach provides protein biomarkers linked to venison meat quality and marked changes in proteins related to oxidoreductase complex in skeletal muscle. SignificanceMeat tenderness and IMF are major concerns in game meat. The proteomic approach demonstrated a strong correlation between proteins and these quality traits, despite the complexity of samples (wild animals hunted at different ages). Our results suggest that several proteins can be biomarkers and provide a molecular insight.

Energy conservation in the gut microbe Methanomassiliicoccus luminyensis is based on membrane‐bound ferredoxin oxidation coupled to heterodisulfide reduction

Methanomassiliicoccus luminyensis was originally isolated from human feces and belongs to the seventh order of methanogens, the Methanomassiliicoccales, which are only distantly related to other methanogenic archaea. The organism forms methane from the reduction of methylamines or methanol using molecular hydrogen as reductant. The energy-conserving system in M.luminyensis is unique and the enzymes involved in this process are not found in this combination in members of the other methanogenic orders. In this context our central question was how the organism is able to generate ATP. Energy transduction was dependent on a membrane-bound ferredoxin: heterodisulfide oxidoreductase composed of reduced ferredoxin as an electron donor, at least one protein in the membrane fraction and the heterodisulfide reductase HdrD, which reduced the electron acceptor CoM-S-S-CoB. Electron transfer of this respiratory chain proceeded with a rate of 145nmol reduced heterodisulfide min-1 ·mg-1 membrane protein. Methanomassiliicoccus luminyensis is the first example of a methanogenic archaeon that does not require Na+ ions for energy conservation. Only protons were used as coupling ions for the generation of the electrochemical ion gradient. The membrane-bound F420 H2 :phenazine oxidoreductase complex (without the electron input module FpoF) probably catalyzed the oxidation of reduced ferredoxin and potentially acted as primary proton pump in this electron transport system. In summary, the energy-conserving system of M.luminyensis possesses features found in the pathways of hydrogenotrophic and methylotrophic/aceticlastic methanogenesis. Consequently, the composition of the enzymes involved in ion translocation across the cytoplasmic membrane is different from all other methanogenic archaea.

Genomic erosion and extensive horizontal gene transfer in gut-associated Acetobacteraceae

BackgroundSymbiotic relationships between animals and bacteria have profound impacts on the evolutionary trajectories of each partner. Animals and gut bacteria engage in a variety of relationships, occasionally persisting over evolutionary timescales. Ants are a diverse group of animals that engage in many types of associations with taxonomically distinct groups of bacterial associates. Here, we bring into culture and characterize two closely-related strains of gut associated Acetobacteraceae (AAB) of the red carpenter ant, Camponotus chromaiodes.ResultsGenome sequencing, assembly, and annotation of both strains delineate stark patterns of genomic erosion and sequence divergence in gut associated AAB. We found widespread horizontal gene transfer (HGT) in these bacterial associates and report elevated gene acquisition associated with energy production and conversion, amino acid and coenzyme transport and metabolism, defense mechanisms, and lysine export. Both strains have acquired the complete NADH-quinone oxidoreductase complex, plausibly from an Enterobacteriaceae origin, likely facilitating energy production under diverse conditions. Conservation of several lysine biosynthetic and salvage pathways and accumulation of lysine export genes via HGT implicate L-lysine supplementation by both strains as a potential functional benefit for the host. These trends are contrasted by genome-wide erosion of several amino acid biosynthetic pathways and pathways in central metabolism. We perform phylogenomic analyses on both strains as well as several free living and host associated AAB. Based on their monophyly and deep divergence from other AAB, these C. chromaiodes gut associates may represent a novel genus. Together, our results demonstrate how extensive horizontal transfer between gut associates along with genome-wide deletions leads to mosaic metabolic pathways. More broadly, these patterns demonstrate that HGT and genomic erosion shape metabolic capabilities of persistent gut associates and influence their genomic evolution.ConclusionsUsing comparative genomics, our study reveals substantial changes in genomic content in persistent associates of the insect gastrointestinal tract and provides evidence for the evolutionary pressures inherent to this environment. We describe patterns of genomic erosion and horizontal acquisition that result in mosaic metabolic pathways. Accordingly, the phylogenetic position of both strains of these associates form a divergent, monophyletic clade sister to gut associates of honey bees and more distantly to Gluconobacter.

Targeting Type IV pili as an antivirulence strategy against invasive meningococcal disease.

Bacterial virulence factors are attractive targets for the development of therapeutics. Type IV pili, which are associated with a remarkable array of properties including motility, the interaction between bacteria and attachment to biotic and abiotic surfaces, represent particularly appealing virulence factor targets. Type IV pili are present in numerous bacterial species and are critical for their pathogenesis. In this study, we report that trifluoperazine and related phenothiazines block functions associated with Type IV pili in different bacterial pathogens, by affecting piliation within minutes. Using Neisseria meningitidis as a paradigm of Gram-negative bacterial pathogens that require Type IV pili for pathogenesis, we show that piliation is sensitive to altered activity of the Na+ pumping NADH-ubiquinone oxidoreductase (Na+-NQR) complex and that these compounds probably altered the establishment of the sodium gradient. In vivo, these compounds exert a strong protective effect. They reduce meningococcal colonization of the human vessels and prevent subsequent vascular dysfunctions, intravascular coagulation and overwhelming inflammation, the hallmarks of invasive meningococcal infections. Finally, they reduce lethality. This work provides a proof of concept that compounds with activity against bacterial Type IV pili could beneficially participate in the treatment of infections caused by Type IV pilus-expressing bacteria.

Composition of Mitochondrial Complex I during the Critical Node of Seed Aging in Oryza sativa

Previous studies have documented mitochondrial dysfunction during the critical node (CN) of rice (Oryza sativa) seed aging, including a decrease in the capacity of NADH dependent O2 consumption. This raises the hypothesis that changes in the activity of NADH:ubiquinone oxidoreductase (complex I) may play a role in seed aging. The composition and activity of complex I was investigated at the CN of aged rice seeds. Using BN-PAGE and SWATH-MS 52 complex I subunits were identified, nineteen for the first time to be experimentally detected in rice. The subunits of the matrix arm (N and Q modules) were reduced in abundance at the CN, in accordance with a reduction in the capacity to oxidise NADH, reducing substrate oxidation and increase ROS accumulation. In contrast, subunits in the P module increased in abundance that contains many mitochondrial encoded subunits. It is proposed that the changes in complex I abundance subunits may indicate a premature re-activation of mitochondrial biogenesis, as evidenced by the increase in mitochondrial encoded subunits. This premature activation of mitochondrial biogenesis may under-pin the decreased viability of aged seeds, as mitochondrial biogenesis is a crucial event in germination to drive growth before autotrophic growth of the seedling is established.

Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis.

The highly oxygen-sensitive hydrogen uptake (Hup) hydrogenase from Dehalococcoides mccartyi forms part of a protein-based respiratory chain coupling hydrogen oxidation with organohalide reduction on the outside of the cell. The HupXSL proteins were previously shown to be synthesized and enzymatically active in Escherichia coli. Here we examined the growth conditions that deliver active Hup enzyme that couples H2 oxidation to benzyl viologen (BV) reduction, and identified host factors important for this process. In a genetic background lacking the three main hydrogenases of E. coli we could show that additional deletion of genes necessary for selenocysteine biosynthesis resulted in inactive Hup enzyme, suggesting requirement of a formate dehydrogenase for Hup activity. Hup activity proved to be dependent on the presence of formate dehydrogenase (Fdh-H), which is typically associated with the H2-evolving formate hydrogenlyase (FHL) complex in the cytoplasm. Further analyses revealed that heterologous Hup activity could be recovered if the genes encoding the ferredoxin-like electron-transfer protein HupX, as well as the related HycB small subunit of Fdh-H were also deleted. These findings indicated that the catalytic HupL and electron-transferring HupS subunits were sufficient for enzyme activity with BV. The presence of the HupX or HycB proteins in the absence of Fdh-H therefore appears to cause inactivation of the HupSL enzyme. This is possibly because HupX or HycB aided transfer of electrons to the quinone pool or other oxidoreductase complexes, thus maintaining the HupSL heterodimer in a continuously oxidized state causing its inactivation. This proposal was supported by the observation that growth under either aerobic or anaerobic respiratory conditions did not yield an active HupSL. These studies thus provide a system to understand the redox sensitivity of this heterologously synthesized hydrogenase.

Frequency of vitamin K oxidoreductase complex subunit-1 (VKORC1) polymorphisms and warfarin dose management in patients with venous thromboembolism.

Warfarin works by inhibiting VKORC1, so polymorphisms of this gene modify the required drug dose. The aim of this study is to examine the relation between therapeutic weekly dose of warfarin and C1173T/G1639A polymorphism of VKORC1 in patients with VTE. Seventy-five patients with VTE were enrolled. Weekly warfarin doses and time (day) to reach therapeutic INR were evaluated retrospectively along with VKORC1-C1173T and G1639A alleles. The mean weekly warfarin dose was lower and time to reach therapeutic INR was shorter in homozygote alleles (AA and TT) (p < 0.05). The multivariate regression model was produced, R2 = 0.05% for age (p = 0.04), R2 = 6% for VKORC1 (p = 0.03), the model for estimating warfarin dose R2 = 17% (p > 0.05). In particular, patients who need overdose of warfarin or whose bleeding score is high, study of these polymorphisms can be considered.

Five decades of research on mitochondrial NADH-quinone oxidoreductase (complex I).

NADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson's and Alzheimer's, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membrane in situ analysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNf and SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping.

Identification and characterization two isoforms of NADH:ubiquinone oxidoreductase from the hyperthermophilic eubacterium Aquifex aeolicus

The NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory chain and the entry point for most electrons. Generally, the bacterial complex I consists of 14 core subunits, homologues of which are also found in complex I of mitochondria. In complex I preparations from the hyperthermophilic bacterium Aquifex aeolicus we have identified 20 partially homologous subunits by combining MALDI-TOF and LILBID mass spectrometry methods. The subunits could be assigned to two different complex I isoforms, named NQOR1 and NQOR2. NQOR1 consists of subunits NuoA2, NuoB, NuoD2, NuoE, NuoF, NuoG, NuoI1, NuoH1, NuoJ1, NuoK1, NuoL1, NuoM1 and NuoN1, with an entire mass of 504.17 kDa. NQOR2 comprises subunits NuoA1, NuoB, NuoD1, NuoE, NuoF, NuoG, NuoH2, NuoI2, NuoJ1, NuoK1, NuoL2, NuoM2 and NuoN2, with a total mass of 523.99 kDa. Three Fe-S clusters could be identified by EPR spectroscopy in a preparation containing predominantly NQOR1. These were tentatively assigned to a binuclear center N1, and two tetranuclear centers, N2 and N4. The redox midpoint potentials of N1 and N2 are −273 mV and −184 mV, respectively. Specific activity assays indicated that NQOR1 from cells grown under low concentrations of oxygen was the more active form. Increasing the concentration of oxygen in the bacterial cultures induced formation of NQOR2 showing the lower specific activity.

Vacuolar Protein Degradation via Autophagy Provides Substrates to Amino Acid Catabolic Pathways as an Adaptive Response to Sugar Starvation in Arabidopsis thaliana.

The vacuolar lytic degradation of proteins releases free amino acids that plants can use instead of sugars for respiratory energy production. Autophagy is a major cellular process leading to the transport of proteins into the vacuole for degradation. Here, we examine the contribution of autophagy to the amino acid metabolism response to sugar starvation in mature leaves of Arabidopsis thaliana. During sugar starvation arising from the exposure of wild-type (WT) plants to darkness, autophagic transport of chloroplast stroma, which contains most of the proteins in a leaf, into the vacuolar lumen was induced within 2 d. During this time, the level of soluble proteins, primarily Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), decreased and the amount of free amino acid increased. In dark-treated autophagy-defective (atg) mutants, the decrease of soluble proteins was suppressed, which resulted in the compromised release of basic amino acids, branched-chain amino acids (BCAAs) and aromatic amino acids. The impairment of BCAA catabolic pathways in the knockout mutants of the electron transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (etfqo) complex and the electron donor protein isovaleryl-CoA dehydrogenase (ivdh) caused a reduced tolerance to dark treatment similar to that in the atg mutants. The enhanced accumulation of BCAAs in the ivdh and etfqo mutants during the dark treatment was reduced by additional autophagy deficiency. These results indicate that vacuolar protein degradation via autophagy serves as an adaptive response to disrupted photosynthesis by providing substrates to amino acid catabolic pathways, including BCAA catabolism mediated by IVDH and ETFQO.

Feeding difficulties, a key feature of the Drosophila NDUFS4 mitochondrial disease model.

ABSTRACTMitochondrial diseases are associated with a wide variety of clinical symptoms and variable degrees of severity. Patients with such diseases generally have a poor prognosis and often an early fatal disease outcome. With an incidence of 1 in 5000 live births and no curative treatments available, relevant animal models to evaluate new therapeutic regimes for mitochondrial diseases are urgently needed. By knocking down ND-18, the unique Drosophila ortholog of NDUFS4, an accessory subunit of the NADH:ubiquinone oxidoreductase (Complex I), we developed and characterized several dNDUFS4 models that recapitulate key features of mitochondrial disease. Like in humans, the dNDUFS4 KD flies display severe feeding difficulties, an aspect of mitochondrial disorders that has so far been largely ignored in animal models. The impact of this finding, and an approach to overcome it, will be discussed in the context of interpreting disease model characterization and intervention studies.This article has an associated First Person interview with the first author of the paper.

Protein Kinase A/CREB Signaling Prevents Adriamycin-Induced Podocyte Apoptosis via Upregulation of Mitochondrial Respiratory Chain Complexes.

Previous work showed that the activation of protein kinase A (PKA) signaling promoted mitochondrial fusion and prevented podocyte apoptosis. The cAMP response element binding protein (CREB) is the main downstream transcription factor of PKA signaling. Here we show that the PKA agonist 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate-cyclic AMP (pCPT-cAMP) prevented the production of adriamycin (ADR)-induced reactive oxygen species and apoptosis in podocytes, which were inhibited by CREB RNA interference (RNAi). The activation of PKA enhanced mitochondrial function and prevented the ADR-induced decrease of mitochondrial respiratory chain complex I subunits, NADH-ubiquinone oxidoreductase complex (ND) 1/3/4 genes, and protein expression. Inhibition of CREB expression alleviated pCPT-cAMP-induced ND3, but not the recovery of ND1/4 protein, in ADR-treated podocytes. In addition, CREB RNAi blocked the pCPT-cAMP-induced increase in ATP and the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-α). The chromatin immunoprecipitation assay showed enrichment of CREB on PGC1-α and ND3 promoters, suggesting that these promoters are CREB targets. In vivo, both an endogenous cAMP activator (isoproterenol) and pCPT-cAMP decreased the albumin/creatinine ratio in mice with ADR nephropathy, reduced glomerular oxidative stress, and retained Wilm's tumor suppressor gene 1 (WT-1)-positive cells in glomeruli. We conclude that the upregulation of mitochondrial respiratory chain proteins played a partial role in the protection of PKA/CREB signaling.

Genome-wide admixture and association study of subclinical atherosclerosis in the Women's Interagency HIV Study (WIHS).

Cardiovascular disease (CVD) is a major comorbidity among HIV-infected individuals. Common carotid artery intima-media thickness (cCIMT) is a valid and reliable subclinical measure of atherosclerosis and is known to predict CVD. We performed genome-wide association (GWA) and admixture analysis among 682 HIV-positive and 288 HIV-negative Black, non-Hispanic women from the Women’s Interagency HIV study (WIHS) cohort using a combined and stratified analysis approach. We found some suggestive associations but none of the SNPs reached genome-wide statistical significance in our GWAS analysis. The top GWAS SNPs were rs2280828 in the region intergenic to mediator complex subunit 30 and exostosin glycosyltransferase 1 (MED30 | EXT1) among all women, rs2907092 in the catenin delta 2 (CTNND2) gene among HIV-positive women, and rs7529733 in the region intergenic to family with sequence similarity 5, member C and regulator of G-protein signaling 18 (FAM5C | RGS18) genes among HIV-negative women. The most significant local European ancestry associations were in the region intergenic to the zinc finger and SCAN domain containing 5D gene and NADH: ubiquinone oxidoreductase complex assembly factor 1 (ZSCAN5D | NDUF1) pseudogene on chromosome 19 among all women, in the region intergenic to vomeronasal 1 receptor 6 pseudogene and zinc finger protein 845 (VN1R6P | ZNF845) gene on chromosome 19 among HIV-positive women, and in the region intergenic to the SEC23-interacting protein and phosphatidic acid phosphatase type 2 domain containing 1A (SEC23IP | PPAPDC1A) genes located on chromosome 10 among HIV-negative women. A number of previously identified SNP associations with cCIMT were also observed and included rs2572204 in the ryanodine receptor 3 (RYR3) and an admixture region in the secretion-regulating guanine nucleotide exchange factor (SERGEF) gene. We report several SNPs and gene regions in the GWAS and admixture analysis, some of which are common across HIV-positive and HIV-negative women as demonstrated using meta-analysis, and also across the two analytic approaches (i.e., GWA and admixture). These findings suggest that local European ancestry plays an important role in genetic associations of cCIMT among black women from WIHS along with other environmental factors that are related to CVD and may also be triggered by HIV. These findings warrant confirmation in independent samples.