Cytochrome B Research Articles

Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii.

As an obligate intracellular parasite, Toxoplasma gondii must import essential nutrients from the host cell into the parasitophorous vacuole. We previously reported that the parasite scavenges cholesterol from host endocytic organelles for incorporation into membranes and storage as cholesteryl esters in lipid droplets. In this study, we have investigated whether Toxoplasma utilizes cholesterol as a precursor for the synthesis of metabolites, such as steroids. In mammalian cells, steroidogenesis occurs in mitochondria and involves membrane-bound type I cytochrome P450 oxidases that are activated through interaction with heme-binding proteins containing a cytochrome b5 domain, such as members of the membrane-associated progesterone receptor (MAPR) family. Our LC-MS targeted lipidomics detect selective classes of hormone steroids in Toxoplasma, with a predominance for anti-inflammatory hydroxypregnenolone species, deoxycorticosterone and dehydroepiandrosterone. The genome of Toxoplasma contains homologs encoding a single type I CYP450 enzyme (we named TgCYP450mt) and a single MAPR (we named TgMAPR). We showed that TgMAPR is a hemoprotein with conserved residues in a heme-binding cytochrome b5 domain. Both TgCYP450 and TgMAPR localize to the mitochondrion and show interactions in in situ proximity ligation assays. Genetic ablation of cyp450mt is not tolerated by Toxoplasma; we therefore engineered a conditional knockout strain and showed that iΔTgCYP450mt parasites exhibit growth impairment in cultured cells. Parasite strains deficient for mapr could be generated; however, ΔTgMAPR parasites suffer from poor global fitness, loss of plasma membrane integrity, aberrant mitochondrial cristae, and an abnormally long S-phase in their cell cycle. Compared to wild-type parasites, iΔTgCYP450mt and ΔTgMAPR lost virulence in mice and metabolomics studies reveal that both mutants have reduced levels of steroids. These observations point to a steroidogenic pathway operational in the mitochondrion of a protozoan that involves an evolutionary conserved TgCYP450mt enzyme and its binding partner TgMAPR.

Cytochrome b561 regulates iron metabolism by activating the Akt/mTOR pathway to promote Breast Cancer Cells proliferation

Breast cancer (BC) is the leading cause of cancer-related mortality in women, necessitating the development of novel therapeutic targets. While cytochrome b561 (CYB561) expression is associated with poor prognosis in BC, the precise role of CYB561 in BC and its potential mechanisms remain unclear. In the present study, we found that CYB561 plays an essential role in BC growth. CYB561 expression was up-regulated in surgically resected cancerous tissues and in six BC cell lines. Lentivirus-mediated CYB561 knockdown in BC cells significantly reduced their proliferation, migration, and invasiveness. CYB561 participates in the regulation of iron metabolism in BC. CYB561 knockdown reduced total iron content, increased ferrous iron content, and down-regulated the expression of proteins associated with iron metabolism (transferrin receptor 1, divalent metal transporter 1, and ferritin heavy chain 1). Conversely, up-regulation of CYB561 through co-incubation with exogenous iron (ferric ammonium citrate) produced contrary outcomes. Additionally, CYB561 activated the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway in BC cells. Down-regulation of CYB561 expression inhibited the Akt/mTOR signaling pathway activity. The application of an mTOR agonist (MHY1485) rescued this negative effect, as well as the inhibitory effect of CYB561 knockdown on cell proliferation. Importantly, the dual mTOR inhibitor MLN0128 (50 nM, 48 h) down-regulated CYB561 expression and the iron metabolism-related proteins transferrin receptor, divalent metal transporter 1, and ferritin heavy chain 1, whereas the mTOR agonist MHY1485 rescued the down-regulation of CYB561 knockdown on iron metabolism-related proteins. We conclude that CYB561 promotes the proliferation of BC cells by regulating iron metabolism through the activation of the Akt/mTOR signaling pathway.

Increased drought resistance in state transition mutants is linked to modified plastoquinone pool redox state.

Identifying traits that exhibit improved drought resistance is highly important to cope with the challenges of predicted climate change. We investigated the response of state transition mutants to drought. Compared with the wild type, state transition mutants were less affected by drought. Photosynthetic parameters in leaves probed by chlorophyll fluorescence confirmed that mutants possess a more reduced plastoquinone (PQ) pool, as expected due to the absence of state transitions. Seedlings of the mutants showed an enhanced growth of the primary root and more lateral root formation. The photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, leading to an oxidised PQ pool, inhibited primary root growth in wild type and mutants, while the cytochrome b6 f complex inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone, leading to a reduced PQ pool, stimulated root growth. A more reduced state of the PQ pool was associated with a slight but significant increase in singlet oxygen production. Singlet oxygen may trigger a, yet unknown, signalling cascade promoting root growth. We propose that photosynthetic mutants with a deregulated ratio of photosystem II to photosystem I activity can provide a novel path for improving crop drought resistance.

Direct interaction between the transmembrane helices stabilize cytochrome P450 2B4 and cytochrome b5 redox complex

The catalytic activity of cytochrome P450 2B4 (CYP2B4) is moderated by its cognate redox partner cytochrome b5 (Cyt-b5). The endoplasmic reticulum (ER) membrane and intermolecular transmembrane (TM) interaction between CYP2B4 and Cyt-b5 regulate the substrate catalysis and the reaction rate. This emphasizes the significance of elucidating the molecular basis of CYP2B4 and Cyt-b5 complexation in a membrane environment to better understand the enzymatic activity of CYP2B4. Our previous solid-state NMR studies revealed the membrane topology of the transmembrane domains of these proteins in the free and complex forms. Here, we show the cross-angle complex formation by the single-pass TM domains of CYP2B4 and Cyt-b5, which is mainly driven by several salt-bridges (E2-R128, R21-D104 and K25-D104), using a multi-microsecond molecular dynamic simulation. Additionally, the leucine-zipper residues (L8, L12, L15, L18 and L19 from CYP2B4) and π-stacking between H23 and F20 residues of CYP2B4 and W110 of Cyt-b5 are identified to stabilize the TM-TM complex in the ER membrane. The simulated tilts of the helices in the free and in the complex are in excellent agreement with solid-state NMR results. The TM-TM packing influences a higher order structural stability when compared to the complex formed by the truncated soluble domains of these two proteins. MM/PBSA based binding free energy estimates nearly 100-fold higher binding affinity (ΔG = -2810.68 ± 696.44 kJ/mol) between the soluble domains of the full-length CYP2B4 and Cyt-b5 when embedded in lipid membrane as compared to the TM-domain-truncated soluble domains (ΔG = -27.406 ± 10.32 kJ/mol). The high-resolution full-length CYP2B4-Cyt-b5 complex structure and its dynamics in a native ER membrane environment reported here could aid in the development of approaches to effectively modulate the drug-metabolism activity of CYP2B4.

Biophysical and molecular characteristics of senescing leaves of two Norway maple varieties differing in anthocyanin content.

Disassembly and degradation of the photosynthetic protein complexes during autumn senescence, a vital step to ensure efficient nutrient relocalization for winter storage, is poorly understood. Concomitantly with the degradation, anthocyanins are often synthesized. However, as to why leaves accumulate red pigments, no consensus exists. One possibility is that anthocyanins protect senescing leaves from excess light. In this study, we investigated the pigment composition, photosynthetic performance, radical production, and degradation of the photosynthetic protein complexes in Norway maple (Acer platanoides) and in its highly pigmented, purple-colored variety (Faassen's black) during autumn senescence, to dissect the possible roles of anthocyanins in photoprotection. Our findings show that senescing Faassen's black was indeed more resistant to Photosystem II (PSII) photoinhibition, presumably due to its high anthocyanin content, than the green maple. However, senescing Faassen's black exhibited low photosynthetic performance, probably due to a poor capacity to repair PSII. Furthermore, an analysis of photosynthetic protein complexes demonstrated that in both maple varieties, the supercomplexes consisting of PSII and its antenna were disassembled first, followed by the degradation of the PSII core, Photosystem I, Cytochrome b6 f, and ATP synthase. Strikingly, the degradation process appeared to proceed faster in Faassen's black, possibly explaining its poor PSII repair capacity. The results suggest that tolerance against PSII photoinhibition may not necessarily translate to a better fitness. Finally, thylakoids isolated from senescing and non-senescing leaves of both maple varieties accumulated very little carbon-centered radicals, suggesting that thylakoids may not be a major source of reactive oxygen species in senescing leaves.

Structure of Chlorella ohadii Photosystem II Reveals Protective Mechanisms against Environmental Stress.

Green alga Chlorella ohadii is known for its ability to carry out photosynthesis under harsh conditions. Using cryogenic electron microscopy (cryoEM), we obtained a high-resolution structure of PSII at 2.72 Å. This structure revealed 64 subunits, which encompassed 386 chlorophylls, 86 carotenoids, four plastoquinones, and several structural lipids. At the luminal side of PSII, a unique subunit arrangement was observed to protect the oxygen-evolving complex. This arrangement involved PsbO (OEE1), PsbP (OEE2), PsbB, and PsbU (a homolog of plant OEE3). PsbU interacted with PsbO, PsbC, and PsbP, thereby stabilizing the shield of the oxygen-evolving complex. Significant changes were also observed at the stromal electron acceptor side. PsbY, identified as a transmembrane helix, was situated alongside PsbF and PsbE, which enclosed cytochrome b559. Supported by the adjacent C-terminal helix of Psb10, these four transmembrane helices formed a bundle that shielded cytochrome b559 from the surrounding solvent. Moreover, the bulk of Psb10 formed a protective cap, which safeguarded the quinone site and likely contributed to the stacking of PSII complexes. Based on our findings, we propose a protective mechanism that prevents QB (plastoquinone B) from becoming fully reduced. This mechanism offers insights into the regulation of electron transfer within PSII.

Biochemical Alterations in Triticale Seedlings Pretreated with Selective Herbicide and Subjected to Drought or Waterlogging Stress.

Waterlogging and drought disrupt crop development and productivity. Triticale is known to be relatively tolerant to different stress factors. In natural conditions, plants are rather subjected to multiple environmental factors. Serrate® (Syngenta) is a systemic selective herbicide suitable for cereal crops such as triticale and wheat to restrain annual grass and broadleaf weeds. Triticale (×Triticosecale Wittm., cv. Rozhen) was grown as soil culture under controlled conditions. Seventeen-day-old plantlets were leaf sprayed with Serrate®. The water stress (drought or waterlogging) was applied after 72 h for 7 days, and then the seedlings were left for recovery. The herbicide does not provoke sharp alterations in the antioxidant state (stress markers level, and antioxidant and xenobiotic-detoxifying enzymes activity). The water stresses and combined treatments enhanced significantly the content of stress markers (malondialdehyde, proline, hydrogen peroxide), non-enzymatic (total phenolics and thiol groups-containing compounds), and enzymatic (activities of superoxide dismutase, catalase, guaiacol peroxidase, glutathione reductase) antioxidants, and xenobiotic-detoxifying enzymes (activities of glutathione S-transferase, NADPH:cytochrome P450 reductase, NADH:cytochrome b5 reductase). These effects were more severely expressed after the drought stress, suggesting that this cultivar is more tolerant to waterlogging than to drought stress.

A possible relationship between the effect of factors on photoactivation of photosystem II depleted of functional Mn and cytochrome b559

The photoassembly of the Mn4CaO5 cluster in Mn-depleted photosystem II preparations (photoactivation) was studied under the influence of oxidants, reductants and pH. New data on the effect of these factors on the photoactivation yield are presented. The presence of the oxidant, ferricyanide, negatively affected the photoactivation yield over the entire concentration range studied (0–1 mM). In contrast to ferricyanide, the addition of the reductant, ferrocyanide, up to 1 mM resulted in an increase in the photoactivation yield. Other reductants either did not significantly affect (diphenylcarbazide) or suppressed (ascorbate) the photoactivation yield. The effect of ferrocyanide on photoactivation were found to be similar dichlorophenolindophenol. Investigation of the photoactivation yield as a function of pH revealed that the maximum yield was observed at pH 6.5 in the presence of ferrocyanide and DCPIP, and at pH 5.5 without additives. In addition, the photoactivation yield at pH 5.5 was the same without and with the addition of ferrocyanide or dichlorophenolindophenol. Although ferricyanide suppressed the photoactivation, the photoactivation yield increased in the presence of ferricyanide by shifting the pH to the acidic region. The samples contained approximately 25 % of the HP cyt b559, which was in the reduced state, as the absorbance at 559 nm was decreased upon addition of ferricyanide and subsequent addition of ferrocyanide returned the spectrum to the baseline. A possible relationship between the effect of factors on the photoactivation and the involvement of cyt b559 in the protection of PSII from oxidative damage on the donor side is discussed.

NdhS interacts with cytochrome b6 f to form a complex in Arabidopsis.

Cyclic electron transport (CET) around photosystem I (PSI) is crucial for photosynthesis to perform photoprotection and sustain the balance of ATP and NADPH. However, the critical component of CET, cyt b6 f complex (cyt b6 f), functions in CET has yet to be understood entirely. In this study, we found that NdhS, a subunit of NADPH dehydrogenase-like (NDH) complex, interacted with cyt b6 f to form a complex in Arabidopsis. This interaction depended on the N-terminal extension of NdhS, which was conserved in eukaryotic plants but defective in prokaryotic algae. The migration of NdhS was much more in cyt b6 f than in PSI-NDH super-complex. Based on these results, we suggested that NdhS and NADP+ oxidoreductase provide a docking domain for the mobile electron carrier ferredoxin to transfer electrons to the plastoquinone pool via cyt b6 f in eukaryotic photosynthesis.

Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium.

Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic E. coli BL-21 and auxotrophic E. coli B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b5 (b5M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic E. coli, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b5M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70-80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.

Osmotic stress-induced localisation switch of CBR1 from mitochondria to the endoplasmic reticulum triggers ATP production via β-oxidation to respond to osmotic shock.

Drought and high salinity are major environmental factors that reduce plant growth and development, leading to loss of plant productivity in agriculture. Under these stress conditions, photosynthesis is greatly suppressed despite the high cellular energy cost of stress response processes. Currently, the process that allows plants to secure the energy required for osmotic stress responses remains elusive. Here, we provide evidence that cytochrome b5 reductase 1 (CBR1), a cytochrome b5 reductase, plays an important role in ATP production in response to NaCl and dehydration stresses. Overexpression and loss of function of CBR1 led to enhanced resistance and sensitivity, respectively, to osmotic stress. Upon exposure to osmotic stress, CBR1 was localised to the endoplasmic reticulum (ER) instead of to mitochondria, where it was localised under normal conditions. Transgenic plants overexpressing ER-targeted CBR1 showed enhanced resistance to osmotic stress. Moreover, CBR1-ER and CBR1-OX plants, had higher levels of ATP and unsaturated fatty acids under osmotic stress. However, these effects were abrogated by thioridazine and 2-deoxy glucose, inhibitors of β-oxidation and glycolysis, respectively. Based on these results, we propose that ER-localised CBR1 triggers ATP production via the production and β-oxidation of polyunsaturated fatty acids under osmotic stress.

Sex-specific metabolic adaptations in transgenic mice overexpressing cytochrome b5 reductase-3

Cytochrome b5 reductase 3 (CYB5R3) activates respiratory metabolism in cellular systems and exerts a prolongevity action in transgenic mice overexpressing this enzyme, mimicking some of the beneficial effects of calorie restriction. The aim of our study was to investigate the role of sex on metabolic adaptations elicited by CYB5R3 overexpression, and how key markers related with mitochondrial function are modulated in skeletal muscle, one of the major contributors to resting energy expenditure. Young CYB5R3 transgenic mice did not exhibit the striking adaptations in carbon metabolism previously detected in older animals. CYB5R3 was efficiently overexpressed and targeted to mitochondria in skeletal muscle from transgenic mice regardless sex. Overexpression significantly elevated NADH in both sexes, although differences were not statistically significant for NAD+, and increased the abundance of cytochrome c and the fission protein DRP-1 in females but not in males. Moreover, while mitochondrial biogenesis and function markers (as TFAM, NRF-1 and cleaved SIRT3) were markedly upregulated by CYB5R3 overexpression in females, a downregulation was observed in males. Ultrastructural changes were also highlighted, with an increase in the number of mitochondria per surface unit, and in the size of intermyofibrillar mitochondria in transgenic females compared with their wild-type controls. Our results support that CYB5R3 overexpression upregulates markers consistent with enhanced mitochondrial biogenesis and function, and increases mitochondrial abundance in skeletal muscle, producing most of these potentially beneficial actions in females.

High light-induced changes in whole-cell proteomic profile and its correlation with the organization of thylakoid super-complex in cyclic electron transport mutants of Chlamydomonas reinhardtii.

Light and nutrients are essential components of photosynthesis. Activating the signaling cascades is critical in starting adaptive processes in response to high light. In this study, we have used wild-type (WT), cyclic electron transport (CET) mutants like Proton Gradient Regulation (PGR) (PGRL1), and PGR5 to elucidate the actual role in regulation and assembly of photosynthetic pigment-protein complexes under high light. Here, we have correlated the biophysical, biochemical, and proteomic approaches to understand the targeted proteins and the organization of thylakoid pigment-protein complexes in the photoacclimation. The proteomic analysis showed that 320 proteins were significantly affected under high light compared to the control and are mainly involved in the photosynthetic electron transport chain, protein synthesis, metabolic process, glycolysis, and proteins involved in cytoskeleton assembly. Additionally, we observed that the cytochrome (Cyt) b6 expression is increased in the pgr5 mutant to regulate proton motive force and ATPase across the thylakoid membrane. The increased Cyt b6 function in pgr5 could be due to the compromised function of chloroplast (cp) ATP synthase subunits for energy generation and photoprotection under high light. Moreover, our proteome data show that the photosystem subunit II (PSBS) protein isoforms (PSBS1 and PSBS2) expressed more than the Light-Harvesting Complex Stress-Related (LHCSR) protein in pgr5 compared to WT and pgrl1 under high light. The immunoblot data shows the photosystem II proteins D1 and D2 accumulated more in pgrl1 and pgr5 than WT under high light. In high light, CP43 and CP47 showed a reduced amount in pgr5 under high light due to changes in chlorophyll and carotenoid content around the PSII protein, which coordinates as a cofactor for efficient energy transfer from the light-harvesting antenna to the photosystem core. BN-PAGE and circular dichroism studies indicate changes in macromolecular assembly and thylakoid super-complexes destacking in pgrl1 and pgr5 due to changes in the pigment-protein complexes under high light. Based on this study, we emphasize that this is an excellent aid in understanding the role of CET mutants in thylakoid protein abundances and super-complex organization under high light.

Pure total flavonoids from citrus alleviate oxidative stress and inflammation in nonalcoholic fatty liver disease by regulating the miR-137-3p/NOXA2/NOX2 pathway

BackgroundNonalcoholic fatty liver disease (NAFLD) has become a global health issue owing to its large disease population and high morbidity. We previously reported that the improvement in oxidative stress (OS) using pure total flavonoids from citrus (PTFC), flavonoids isolated from the peel of Citrus changshan-huyou Y.B. Chan, is a crucial strategy for NAFLD treatment. However, OS-associated intervention pathways in NAFLD remain unclear. MethodsIn this study, we used microRNA (miR)- and mRNA-sequencing to identify the pathway by which PTFC improve OS in NAFLD. Clinical data, mimic/inhibitor assays, and a dual-luciferase reporter assay were selected to verify the regulatory relationships of this pathway. Moreover, in vivo and in vitro experiments were used to confime the regulatory effect of PTFC on this pathway. ResultsmiR-seq, mRNA-seq, and bioinformatics analyses revealed that the miR-137–3p/neutrophil cytosolic factor 2 (NCF2, also known as NOXA2)/cytochrome b-245 beta chain (CYBB, also known as NOX2) pathway may be a target pathway for PTFC to improve OS and NAFLD. Additionally, bivariate logistic regression analysis combining the serum and clinical data of patients revealed NOX2 and NOXA2 as risk factors and total antioxidant capacity (indicator of OS level) as a protective factor for NAFLD. miR-137–3p mimic/inhibitor assays revealed that the upregulation of miR-137–3p is vital for improving cellular steatosis, OS, and inflammation. Dual-luciferase reporter assay confirmed that NOXA2 acts as an miR-137–3p sponge. These results co-determined that miR-137–3p/NOXA2/NOX2 is an essential pathway involved in NAFLD pathogenesis, including lipid accumulation, OS, and inflammation. In vivo and in vitro experiments further confirmed that the miR-137–3p/NOXA2/NOX2 pathway is regulated by PTFC. ConclusionPTFC alleviates OS and inflammation in NAFLD by regulating the miR-137–3p/NOXA2/NOX2 pathway.

A Novel Pathway of Functional microRNA Uptake and Mitochondria Delivery.

Extracellular microRNAs (miRNAs) play a critical role in horizontal gene regulation. Uptake of extracellular miRNAs by recipient cells and their intracellular transport, however, remains elusive. Here RNA phase separation is shown as a novel pathway of miRNA uptake. In the presence of serum, synthetic miRNAs rapidly self-assembly into ≈110nm discrete nanoparticles, which enable miRNAs' entry into different cells. Depleting serum cationic proteins prevents the formation of such nanoparticles and thus blocks miRNA uptake. Different from lipofectamine-mediated miRNA transfection in which majority of miRNAs are accumulated in lysosomes of transfected cells, nanoparticles-mediated miRNA uptake predominantly delivers miRNAs into mitochondria in a polyribonucleotide nucleotidyltransferase 1(PNPT1)-dependent manner. Functional assays further show that the internalized miR-21 via miRNA phase separation enhances mitochondrial translation of cytochrome b (CYB), leading to increase in adenosine triphosphate (ATP) and reactive oxygenspecies (ROS) reduction in HEK293T cells. The findings thus reveal a previously unrecognized mechanism for uptake and delivery functional extracellular miRNAs into mitochondria.

Far-red photosynthesis: Two charge separation pathways exist in plant Photosystem II reaction center

An alternative charge separation pathway in Photosystem II under the far-red light was proposed by us on the basis of electron transfer properties at 295 K and 5 K. Here we extend these studies to the temperature range of 77–295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of Cytochrome b559 and ChlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730–750 nm) spectral regions. Temperature dependence of the S2 state signal induction after single flash at 730–750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data indicate that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in Photosystem II with localization of hole on the ChlD1 molecule.

A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Oxidative Metabolism Genes in Ovarian Neoplasms

BACKGROUND: Reactive oxygen species play important and ambiguous role in carcinogenesis, and local oxidative metabolism may differ significantly from systemic metabolism and determine the processes occurring in tumor tissues.
 AIM: This study aimed to examine the expressions of key oxidative metabolism genes, particularly CYB5R, POR, NOX4, SOD1, NF-B, and NRF2, in ovarian neoplasm tissues, and determine cytochrome b5 reductase and cytochrome P450 reductase activity, blood neutrophil activity, and antioxidant indices in the blood plasma and peritoneal fluid.
 MATERIALS AND METHODS: The study included two groups of patients: a study group (n = 10) with ovarian adenocarcinoma and a comparison group (n = 6) with benign ovarian neoplasms. The expressions of CYB5R1, CYB5R2/R4, CYB5R3, POR, BIRC3, NOX4, NRF2, NF-B, SOD1, HMOX1, and BCL2 genes, cytochrome b5 reductase, and cytochrome P450 reductase activity, oxidative activity of blood neutrophils, and antioxidant potential of plasma and peritoneal fluid were evaluated in these two groups of women.
 RESULTS: The expression levels of CYB5R3 and BCL2 were significantly lower in adenocarcinoma tissues. The activities of cytochrome b5 reductase and cytochrome P450 reductase increased in the group with adenocarcinoma. On average, the activity of blood neutrophils corresponded to the reference values. For blood plasma, the antioxidant capacity were not different, whereas the antioxidant capacity in the peritoneal fluid increased approximately twofold in ovarian cancer.
 CONCLUSIONS: Significantly increased cytochrome b5 reductase activity in adenocarcinoma tissues may be a response to intracellular oxidative stress, whereas CYB5R3 gene expression may be reduced by a negative feedback mechanism. The activities of cytochrome P450 reductase and its gene change to a lesser extent in the presence of ovarian adenocarcinoma. The oxidative balance in the blood and peritoneal fluid correlated with the tissue expressions of NF-B and NRF2.

#2853 MITOCHONDRIAL DNA DEREGULATED PATTERN PARALLELS INFLAMMATION IN EARLY DIABETIC KIDNEY DISEASE OF TYPE 2 DIABETES MELLITUS PATIENTS

Abstract Background and Aims Mitochondrial DNA (mtDNA) abnormalities, as well as complex inflammatory processes involved in the pathogenesis of DKD may be detected early in the course of DKD. The aim of the study was to evaluate a potential relation of mtDNA modifications in blood and urine with podocyte injury and proximal tubule (PT) dysfunction in early DKD of type 2 diabetes mellitus (DM) patients. The hypothesis according to which mtDNA changes may be related to a specific inflammatory response in normoalbuminuric DKD of type 2 DM patients was also questioned. Method A total of 150 patients [52 patients with normoalbuminuria, 48 patients with microalbuminuria, and 50 patients with macroalbuminuria)] and 30 age- and gender-matched healthy controls were enrolled in this case series study. All patients were assessed concerning urinary albumin/creatinine ratio (UACR), biomarkers of podocyte damage (synaptopodin, podocalyxin) and of PT dysfunction (kidney injury molecule-1-KIM-1, N-acetyl-D-glucosaminidase-NAG), serum and urinary interleukins (IL-17A, IL-18, IL-10), and eGFR (creatinine-cystatin C). MtDNA-CN and nuclear DNA (nDNA) was quantified in peripheral blood and urine by qRT-PCR (ABI 7900-HT–Applied BioSystem). TaqMan assays were utilized for the assessment of cytochrome b (CYTB) gene, subunit 2 of NADH dehydrogenase (ND2), and of beta 2 microglobulin nuclear gene (B2M). mtDNA-CN was defined as the ratio of the number of mtDNA/nDNA copies, through analysis of the CYTB/B2M and ND2/B2M ratio. DNA standards were created by plasmidial cloning vectors. Cloning was verified by PCR sequencing and validation. Results Serum mtDNA was decreased, while urinary mtDNA was increased in all study groups as compared to healthy controls. In univariable linear analysis, serum mtDNA correlated directly with eGFR and negatively with UACR, urinary synaptopodin, urinary podocalyxin, urinary KIM-1, and urinary NAG. Also, serum mtDNA correlated negatively with serum IL-17A and urinary IL-17A, and with serum IL-18 and urinary IL-18, and directly with serum IL-10 and urinary IL-10. Urinary mtDNA correlated directly with UACR, synaptopodin, podocalyxin, KIM-1, NAG, IL-17A, and IL-18, and showed an inverse correlation with IL-10, and eGFR. Multivariable linear analysis provided models in which serum mtDNA correlated directly with IL-10, and indirectly with UACR, IL-17A, and KIM-1 (R2 = 0.626; p = 0.0001). Urinary mtDNA correlated directly with UACR, podocalyxin, IL-18, and NAG, and negatively with IL-10 and eGFR (R2 = 0.631; p = 0.0001). This data suggests that increased urinary mtDNA levels may be attributed to increased filtration of circulating DNA and that this may derive from filtered circulating mtDNA due to lesions to the glomerular and tubular structures. The correlations of mtDNA with proinflammatory cytokines IL-17A and IL-18, as well as with the anti-inflammatory cytokine IL-10 could explain the association of these cytokines with mtDNA variations and with the expression of podocyte damage and PT dysfunction biomarkers in all studied groups, even in normoalbuminuric type 2 DM patients. Conclusion MtDNA abnormalities and inflammatory processes occur early in the course of type 2 DM. MtDNA displays a specific profile in relation to inflammation both at podocyte and tubular level in normoalbuminuric patients with type 2 DM. These observations are independent of renal function decline.

Role of yceA-cybB-yceB operon in oxidative stress tolerance, swimming motility and antibiotic susceptibility of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is ubiquitous in the environment and is an important MDR opportunistic pathogen. Oxidative stress is an inevitable challenge to an aerobic bacterium. Accordingly, S. maltophilia has many capabilities to face variable oxidative stress. Some of the oxidative stress alleviation systems cross-protect bacteria from antibiotics. In our recent RNA-sequencing transcriptome analysis, we documented the increased expression of a three-gene cluster, yceA-cybB-yceB, in the presence of hydrogen peroxide (H2O2). The YceI-like, cytochrome b561 and YceI-like proteins encoded by yceA, cybB and yceB are located in the cytoplasm, inner membrane and periplasm, respectively. To characterize the role of the yceA-cybB-yceB operon of S. maltophilia in oxidative stress tolerance, swimming motility and antibiotic susceptibility. The presence of the yceA-cybB-yceB operon was verified by RT-PCR. The functions of this operon were revealed by in-frame deletion mutant construction and complementation assay. Expression of the yceA-cybB-yceB operon was assessed by quantitative RT-PCR. The yceA, cybB and yceB genes form an operon. Loss of function of the yceA-cybB-yceB operon compromised menadione tolerance, enhanced swimming motility and increased susceptibility to fluoroquinolone and β-lactam antibiotics. The expression of the yceA-cybB-yceB operon was up-regulated by oxidative stress, such as H2O2 and superoxide, and not impacted by antibiotics, such as fluoroquinolone and β-lactams. The evidence strongly supports the view that the physiological function of the yceA-cybB-yceB operon is to alleviate oxidative stress. The operon provides an additional example that oxidative stress alleviation systems can cross-protect S. maltophilia from antibiotics.