Synthesis Of Cytochrome Research Articles

P53 Orchestrates Cancer Metabolism: Unveiling Strategies to Reverse the Warburg Effect

Cancer cells exhibit significant alterations in their metabolism, characterised by a reduction in oxidative phosphorylation (OXPHOS) and an increased reliance on glycolysis, even in the presence of oxygen. This metabolic shift, known as the Warburg effect, is pivotal in fuelling cancer’s uncontrolled growth, invasion, and therapeutic resistance. While dysregulation of many genes contributes to this metabolic shift, the tumour suppressor gene p53 emerges as a master player. Yet, the molecular mechanisms remain elusive. This study introduces a comprehensive mathematical model, integrating essential p53 targets, offering insights into how p53 orchestrates its targets to redirect cancer metabolism towards an OXPHOS-dominant state. Simulation outcomes align closely with experimental data comparing glucose metabolism in colon cancer cells with wild-type and mutated p53. Additionally, our findings reveal the dynamic capability of elevated p53 activation to fully reverse the Warburg effect, highlighting the significance of its activity levels not just in triggering apoptosis (programmed cell death) post-chemotherapy but also in modifying the metabolic pathways implicated in treatment resistance. In scenarios of p53 mutations, our analysis suggests targeting glycolysis-instigating signalling pathways as an alternative strategy, whereas targeting solely synthesis of cytochrome c oxidase 2 (SCO2) does support mitochondrial respiration but may not effectively suppress the glycolysis pathway, potentially boosting the energy production and cancer cell viability.

Genetic Code Expansion in Shewanella oneidensis MR-1 Allows Site-Specific Incorporation of Bioorthogonal Functional Groups into a c-Type Cytochrome.

Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the understanding and modulation of biological systems and engineer new biotechnology. Here, we report the development of efficient methods for site-specific incorporation of structurally diverse noncanonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated bioorthogonal functional groups could be used for efficient site-selective labeling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.

Iron-mediated DAMO–anammox process: Revealing the mechanism of electron transfer

The nitrate denitrifying anaerobic methane oxidation–anaerobic ammonia oxidation (DAMO–anammox) can accomplish nitrogen removal and methane (CH4) reduction. This process greatly contributes to carbon emission mitigation and carbon neutrality. In this study, we investigated the electron transfer process of functional microorganisms in the iron-mediated DAMO–anammox system. Fe3+ could be bound to several functional groups (-CH3, COO-, –CH) in extracellular polymeric substance (EPS), and the functional groups bound were different at different iron concentration. Fe3+ underwent reduction reactions to produce Fe2+. Most Fe3+ and Fe2+ react with microorganisms and formed chelates with EPS. Three-dimensional fluorescence spectra showed that Fe3+ affected the secretion of tyrosine and tryptophan, which were essential for cytochrome synthesis. The presence of Fe3+ accelerated c-type cytochrome-mediated extracellular electron transfer (EET), and when more Fe3+ existed, the more cytochrome C expressed. DAMO archaea (M. nitroreducens) in the system exhibited a high positive correlation with the functional genes (resa and ccda) for cytochrome c synthesis. Some denitrifying microorganisms showed positive correlations with the abundance of riboflavin. This finding showed that riboflavin secreted by functional microorganisms acted as an electron shuttle. In addition, DAMO archaea were positively correlated with the hair synthesis gene pily1, which indicated that direct interspecies electron transfer (DIET) may exist in the iron-mediated DAMO–anammox system.

Integrative transcriptome and whole-genome bisulfite sequencing analyses of a temperature-sensitive albino tea plant cultivar.

Green tea made from albino buds and leaves has a strong umami taste and aroma. The cultivar 'Zhonghuang 2' (ZH2, Camellia sinensis) is a natural mutant with young shoots that are yellow in spring and green or yellow-green in summer. However, the mechanism of leaf color change remains unclear. Here, we found that young shoots of ZH2 were yellow at low temperature (LT) and green at high temperature (HT), indicating that ZH2 is a temperature-sensitive cultivar. Transmission electron microscopy analysis showed that the grana in the chloroplasts of young shoots grown at LT were poorly stacked, which caused a lack of photoreactions and chlorophyll. RNA-seq results showed 1279 genes differentially expressed in the young shoots grown at LT compared with those at HT, including genes related to cytochrome synthesis, chloroplast development, photosynthesis, and DNA methylation. A whole-genome bisulfite sequencing assay revealed that the dynamics of DNA methylation levels in the CG, CHG, and CHH contexts decreased under LT, and the change was most obvious in the CHH context. Furthermore, 72 genes showed significant changes in both expression and DNA methylation levels, and most of them were related to cytochrome synthesis, chloroplast development, photosynthesis, transcription factors, and signaling pathways. These results demonstrate that DNA methylation is involved in the LT-regulated albino processes of ZH2. Changes in DNA methylation levels were associated with changes in gene expression levels, affecting the structure and function of chloroplasts, which may have a phenotypic impact on shoot and leaf color.

Identifying Genetic Signatures Associated with Oncogene-Induced Replication Stress in Osteosarcoma and Screening for Potential Targeted Drugs.

Osteosarcoma is the most common type of primary malignant bone tumor. Due to the lack of selectivity and sensitivity of chemotherapy drugs to tumor cells, coupled with the use of large doses, chemotherapy drugs often have systemic toxicity. The use of modern sequencing technology to screen tumor markers in a large number of tumor samples is a common method for screening highly specific and selective anti-tumor drugs. This study aims to identify potential biomarkers using the latest reported gene expression signatures of oncogene-induced replication stress (ORS) in aggressive cancers, and potential anti-osteosarcoma drugs were screened in different drug databases. In this study, we obtained 89 osteosarcoma-related samples in the TARGET database, all of which included survival information. According to the median expression of each of six reported ORS gene markers (NAT10/DDX27/ZNF48/C8ORF33/MOCS3/MPP6), we divided 89 osteosarcoma gene expression datasets into a high expression group and a low expression group and then performed a differentially expressed gene (DEG) analysis. The coexisting genes of 6 groups of DEGs were used as replication stress-related genes (RSGs) of osteosarcoma. Then, key RSGs were screened using LASSO regression, a Cox risk proportional regression prognostic model and a tenfold cross-validation test. GSE21257 datasets collected from the Gene Expression Omnibus (GEO) database were used to verify the prognostic model. The final key RSGs selected were used in the L1000PWD and DGIdb databases to mine potential drugs. After further validation by the prognostic model, we identified seven genes associated with ORS in osteosarcoma as key RSGs, including transcription factor 7 like 2 (TCF7L2), solute carrier family 27 member 4 (SLC27A4), proprotein convertase subtilisin/kexin type 5 (PCSK5), nucleolar protein 6 (NOL6), coiled-coil-coil-coil-coil-helix domain containing 4 (CHCHD4), eukaryotic translation initiation factor 3 subunit B (EIF3B), and synthesis of cytochrome C oxidase 1 (SCO1). Then, we screened the seven key RSGs in two drug databases and found six potential anti-osteosarcoma drugs (D GIdb database: repaglinide, tacrolimus, sirolimus, cyclosporine, and hydrochlorothiazide; L1000PWD database: the small molecule VU-0365117-1). Seven RSGs (TCF7L2, SLC27A4, PCSK5, NOL6, CHCHD4, EIF3B, and SCO1) may be associated with the ORS gene signatures in osteosarcoma. Repaglinide, tacrolimus, sirolimus, cyclosporine, hydrochlorothiazide and the small molecule VU-0365117-1 are potential therapeutic drugs for osteosarcoma.

Counteracting Colon Cancer by Inhibiting Mitochondrial Respiration and Glycolysis with a Selective PKCδ Activator.

Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.

THE ACTIVITY OF HEME OXYGENASE AND δ-AMINOLEVULINATE SYNTHASE IN THE RAT KIDNEY UNDER CONDITIONS OF TOXIC INJURY WITH ACETAMINOPHEN

The aim of this work was to study the activity of δ-aminolevulinate synthase, heme oxygenase and the content of mitochondrial cytochromes in the rat kidney conditions of toxic damage with acetaminophen against the background of protein deficiency. δ-Aminolevulinate synthase activity was determined spectrophotometrically taking into account the molar extinction coefficient of 0.023·103 mol-1·cm-1, heme oxygenase activity – by the amount of bilirubin formed and expressed in nanomoles per 1 mg of protein per 1 minute. The determination of the contents of cytochromes a+a3, b, c and c1 in the suspension of isolated mitochondria was carried out by the difference spectra between the reduced and oxidized states in the region of their spectral maxima. The study was conducted on 4 groups of animals: Group I - control animals (K); II – rats kept on a low-protein diet (LPD); ІІІ – animals with acetaminophen-induced liver injury receiving complete ration (AII); IV – animals with acetaminophen-induced liver injury that were previously maintained on semi-synthetic low-protein ration (LPD/AII). It was established that under the conditions of consumption of a low-protein diet, there are no significant changes in the activities of heme oxygenase and δ-aminolevulinate synthase, but a decrease in the content of mitochondrial cytochromes a+a3, b, c, c1 is observed, which probably indicates the critical role of providing protein in the diet for the synthesis of cytochromes. At the same time, under the conditions of toxic damage by acetaminophen, regardless of whether the ration is provided with protein, there is a decrease in the activity of δ-aminolevulinate synthase by an average of 1.6 times with a simultaneous increase in the activity of heme oxygenase by approximately 1.5 times compared to the control values, which indicates an increase in the processes of heme catabolism on against the background of slowing down of its synthesis under the studied conditions. The increase in the activity of heme oxygenase against the background of the decrease in the activity of δ-aminolevulinate synthase can be considered as one of the possible mechanisms of the established decrease in the content of mitochondrial cytochromes under the conditions of acetaminophen intoxication, which in turn may further lead to a violation of the activity of the enzymes of the cytochrome part of the respiratory chain. The obtained results regarding the content of cytochromes, the activities of key enzymes of heme metabolism in the kidneys can be considered as prerequisites for deepening the energy imbalance under the conditions of toxic damage to the kidneys by acetaminophen.

Fe3+ opposes the 1,25(OH)2D3-induced calcium transport across intestinal epithelium-like Caco-2 monolayer in the presence or absence of ascorbic acid.

Although iron is an essential element for hemoglobin and cytochrome synthesis, excessive intestinal iron absorption—as seen in dietary iron supplementation and hereditary disease called thalassemia—could interfere with transepithelial transport of calcium across the intestinal mucosa. The underlying cellular mechanism of iron-induced decrease in intestinal calcium absorption remains elusive, but it has been hypothesized that excess iron probably negates the actions of 1,25-dihydroxyvitamin D [1,25(OH)2D3]. Herein, we exposed the 1,25(OH)2D3-treated epithelium-like Caco-2 monolayer to FeCl3 to demonstrate the inhibitory effect of ferric ion on 1,25(OH)2D3-induced transepithelial calcium transport. We found that a 24-h exposure to FeCl3 on the apical side significantly decreased calcium transport, while increasing the transepithelial resistance (TER) in 1,25(OH)2D3-treated monolayer. The inhibitory action of FeCl3 was considered rapid since 60-min exposure was sufficient to block the 1,25(OH)2D3-induced decrease in TER and increase in calcium flux. Interestingly, FeCl3 did not affect the baseline calcium transport in the absence of 1,25(OH)2D3 treatment. Furthermore, although ascorbic acid is often administered to maximize calcium solubility and to enhance intestinal calcium absorption, it apparently had no effect on calcium transport across the FeCl3- and 1,25(OH)2D3-treated Caco-2 monolayer. In conclusion, apical exposure to ferric ion appeared to negate the 1,25(OH)2D3-stimulated calcium transport across the intestinal epithelium. The present finding has, therefore, provided important information for development of calcium and iron supplement products and treatment protocol for specific groups of individuals, such as thalassemia patients and pregnant women.

Transcriptome Analysis to Identify Crucial Genes for Reinforcing Flavins-Mediated Extracellular Electron Transfer in Shewanella oneidensis.

Flavins serve as the electron mediators in Shewanella oneidensis, determining the extracellular electron transfer (EET) rate. Currently, metabolic engineering of flavins biosynthetic pathway has been studied for improving EET. However, the cellular response triggered by flavins that contribute to EET remains to be elucidated. In this study, the riboflavin-overproducing strain C5 (expressing the flavins synthetic genes in plasmid PYYDT) and the PYYDT strain (harboring the empty plasmid PYYDT) in the microbial fuel cells are applied for comparative transcriptomic analyses to investigate beneficial gene targets that could improve EET. From the differentially expressed genes, we select the significantly upregulated and downregulated genes for inverse engineering in S. oneidensis. The results show that overexpression of ahpC and ccpA, and inactivation of pubA, putB, and tonB are able to improve the EET capability. Combinatorial modulation of these five genes results in the recombinant strain CM4, achieving the maximum power density of 651.78 ± 124.60 mW/m2, 1.97 folds of the parental strain. These genes modulation is speculated to reduce the ROS damage and to promote cytochrome synthesis and heme accumulation, which coherently enhance EET. Our findings facilitate in-depth understanding of the mechanism of flavins-mediated EET and provide new insights in promoting EET of S. oneidensis for electricity generation.

Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.

Mutations in genes encoding cytochrome c oxidase (mitochondrial complex IV) subunits and assembly factors [e.g., synthesis of cytochrome c oxidase 2 (SCO2)] are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumor suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the insulin‐like growth factor 1 receptor (IGF1R)/AKT axis with paradoxical downregulation of mTOR signaling, which was accompanied by increased AMP‐activated kinase activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

Expression of SCO1 and SCO2 after form-deprivation myopia in Guinea pigs.

The retina is a highly energy-consuming tissue associated with visual development, and the reduced quality of retinal imaging can be related to myopia. Synthesis of cytochrome c oxidase 1 (SCO1) and synthesis of cytochrome c oxidase 2 (SCO2) are involved in ATP (adenosine triphosphate) synthesis and energy metabolism. This study aimed to observe the morphologic changes and investigate the expression of SCO1 and SCO2 induced by form-deprivation myopia (FDM) in the retina and sclera of guinea pigs. Thirty-six 3-week-old male guinea pigs were randomly assigned to one of two groups: (1) the model group (n = 18), in which the right eyes were covered by a thin opaque balloon as FDM group, and the left eyes were uncovered and served as the contralateral control group; (2) the blank control group (n = 18), in which bilateral eye received no manipulation. Eyeballs were enucleated for histological analysis. The retina and sclera of the guinea pigs were separated to determine the protein and mRNA expression levels of SCO1 and SCO2, respectively. After four weeks of form deprivation (FD), the refractive degree and axial length increased significantly (P < 0.001). The retinal and scleral tissues were moderately thinner, and the ganglion cells and the cells of inner and outer nuclear layers in the retina became fewer. Compared with the contralateral control group (P < 0.001) and the blank control group (P < 0.001), the collagen content of the sclera became less in the FDM group. The protein and mRNA expression levels of SCO1 and SCO2 in the FDM group were significantly lower than those in the contralateral control group and the blank control group (P < 0.05). The morphologies of the retina and sclera were changed, and the expression of SCO1 and SCO2 at the protein and transcription levels was significantly reduced in the FDM group. Given these changes, SCO1 and SCO2 genes may be involved in myopic progression.

The function of Scox in glial cells is essential for locomotive ability in Drosophila

Synthesis of cytochrome c oxidase (Scox) is a Drosophila homolog of human SCO2 encoding a metallochaperone that transports copper to cytochrome c, and is an essential protein for the assembly of cytochrome c oxidase in the mitochondrial respiratory chain complex. SCO2 is highly conserved in a wide variety of species across prokaryotes and eukaryotes, and mutations in SCO2 are known to cause mitochondrial diseases such as fatal infantile cardioencephalomyopathy, Leigh syndrome, and Charcot-Marie-Tooth disease, a neurodegenerative disorder. These diseases have a common symptom of locomotive dysfunction. However, the mechanisms of their pathogenesis remain unknown, and no fundamental medications or therapies have been established for these diseases. In this study, we demonstrated that the glial cell-specific knockdown of Scox perturbs the mitochondrial morphology and function, and locomotive behavior in Drosophila. In addition, the morphology and function of synapses were impaired in the glial cell-specific Scox knockdown. Furthermore, Scox knockdown in ensheathing glia, one type of glial cell in Drosophila, resulted in larval and adult locomotive dysfunction. This study suggests that the impairment of Scox in glial cells in the Drosophila CNS mimics the pathological phenotypes observed by mutations in the SCO2 gene in humans.

Loss of Functional SCO2 Attenuates Oxidative Stress in Diabetic Kidney Disease.

Increased oxidative stress in glomerular endothelial cells (GEnCs) contributes to early diabetic kidney disease (DKD). While mitochondrial respiratory complex IV activity is reduced in DKD, it remains unclear whether this is a driver or a consequence of oxidative stress in GEnCs. Synthesis of cytochrome C oxidase 2 (SCO2), a key metallochaperone in the electron transport chain, is critical to the biogenesis and assembly of subunits required for functional respiratory complex IV activity. Here, we investigated the effects of Sco2 hypomorphs (Sco2 KO/KI , Sco2 KI/KI ), with a functional loss of SCO2, in the progression of DKD using a murine model of Type II Diabetes Mellitus, db/db mice. Diabetic Sco2 KO/KI and Sco2 KI/KI hypomorphs exhibited a reduction in complex IV activity, but an improvement in albuminuria, serum creatinine, and histomorphometric evidence of early DKD as compared to db/db mice. Single-nucleus RNA sequencing with gene set enrichment analysis of differentially expressed genes in the endothelial cluster of Sco2 KO/KI ;db/db mice demonstrated an increase in genes involved in VEGF-VEGFR2 signaling and reduced oxidative stress as compared to db/db mice. These data suggest that reduced complex IV activity due to a loss of functional SCO2 might be protective in GEnCs in early DKD.

Prospective antibacterial inhibition of c-type cytochrome synthesis.

Sutherland et al. recently reported the in vitro inhibition of Helicobacter hepaticus cytochrome c (cyt c) synthase, which may inactivate c-type cytochromes in this and in other pathogenic bacteria. Here, I discuss the impact of this work, which identifies this conserved synthase as a potential antibacterial target.

Phylogenetic Analysis of Strawberry, Fragaria x ananassa (Rosaceae: Fragaria) using Cytochrome C Heme Attachment Protein (ccsA) Gene

Phylogenetic relationships and genetic diversity were inferred using nucleotide sequences of chloroplast ccsAgene (cytochrome c heme attachment protein, for c-type cytochrome synthesis) of strawberry, F. x ananassa and other Fragaria species. Genomic DNA was isolated from fresh and green leaves of strawberry cultivar ‘Chandler’ seedlings. The ccsA gene sequence was amplified and isolated by using gene specific primers designed from consensus sequences of Fragaria species. After sequencing, the isolated ccsA gene sequence was corrected using BioEdit programs. Then ccsA nucleotide sequence isolated from cultivated strawberry (F. x ananassa) cultivar ‘Chandler’ was deposited in GeneBank at NCBI with accession number MK872805.1. Based on the BLASTn hit results, two separate datasets were prepared, one for Fragaria genus and other for Rosaceae family species to study the diversity analyses. MEGA 7.0 program was then used for phylogenetic analyzes and the detection of evolutionary genetic divergence between the species. For Fragaria genus which also included cultivated strawberry (F. x ananassa) ccsA gene sequence, the mean nucleotide composition was estimated as 29.3% A, 37.8% T, 15.5% C and 17.4% G. The lowest divergence distance i.e., 0.00 was estimated for Fragaris species, while the highest distance 1.00 was observed only for Fragaria nipponica (KY769125.1). Overall, the molecular phylogenetic analysis revealed that no genetic variation was observed in Fragaria species that was due to the highly conserved chloroplast nucleotide sequences of ccsA gene during evolution. However, the ccsA nucleotide sequences can be useful to study intra genus evolutionary relationship between Rosaceae family members.

Complete chloroplast genome sequencing of Job’s tears (Coix L.): genome structure, comparative analysis, and phylogenetic relationships

Job’s tears, also known as adlay, is a valuable plant that has commonly been used in traditional Chinese medicine, as well as an edible food. Due to the lack of knowledge of its genetics and gaps in its evolutionary analysis, breeding of adlay has been hindered. Here, we report five complete chloroplast genomes of various species and varieties in the genus by Illumina sequencing, while their genome structure, comparative analysis, and phylogenetic relationships were conducted. Genome sizes ranged from 140,860 to 140,864 bp in length, GC contents were 38.43%, and genome architecture was of a typical quadripartite structure. We annotated 82～83 protein-coding genes and 46～47 non-coding RNA genes in each genome and they functionally associated with self-replication, photosynthesis, cytochrome synthesis and other unknown functions. Three codons that encoded tryptophan, arginine and leucine were used frequently at rates of 41.42, 37.98, and 32.28% respectively. The preferred codons consistently ended with A or T. A total of 146 simple sequence repeats (SSR), 9 insertions and deletions (InDels) and 143 single nucleotide polymorphisms (SNPs) were observed among genomes. The InDel and SNP variations were mostly distributed in intergenic regions. It confirmed that Coix, Sorghum, Saccharum, Zea, Tripsacum and Saccharum were closely genera and the genetic distance of Sorghum to Coix was closer than Zea to Coix. These results give us more insight into the evolution of Coix in a wide range of evolutionary studies.

Abstract A20: Investigating the role of SCO2 in the metabolic adaptation of cancer cells

Abstract SCO2 (synthesis of cytochrome c oxidase 2) is essential for the assembly and functioning of mitochondrial cytochrome c oxidase (COX) complex (IV). Mutations in the SCO2 gene have been reported to cause COX deficiency and result in fatal infantile cardioencephalomyopathy. Furthermore, the tumor suppressor protein p53 has been shown to mediate its effects on mitochondrial respiration via SCO2. SCO2-/- cells also exhibit increased intracellular oxygen and nicotinamide adenine dinucleotide (NADH) levels, leading to increased reactive oxygen species (ROS) and oxidative DNA damage. However, little is known regarding other metabolic adaptations triggered in response to SCO2 loss. To this end, metabolic profiling of wild-type (WT) and SCO2-/- colon cancer HCT116 cells was carried out using GC/MS. The steady-state levels of most of the glycolytic intermediates were higher in SCO2-/- as compared to WT cells with the exception of pyruvate. Moreover, an asymmetry was observed in the levels of the different tricarboxylic acid (TCA) cycle intermediates when contrasting WT and SCO2-/- cells. Citrate, malate, and alpha-ketoglutarate were elevated in WT relative to SCO2-/- HCT116 cells. In turn, succinate and 2-hydroxyglutarate were higher in SCO2-/- as compared to WT cells. Furthermore, the level of aspartate and asparagine was observed to be lower in the SCO2-/- HCT 116 compared to the WT HCT 116 cells, which is most likely to stem from the defects in oxidative phosphorylation caused by SCO2 loss. Stable isotope tracer analysis using 13C labeled glucose was done to monitor the flux of carbon atoms derived from glucose. As expected, SCO2-/- cells have minimal flux of carbon into the TCA cycle via acetyl CoA production due to the dysfunctional electron transport chain compared to the WT cells. However, SCO2-/- cells are still able to biosynthesize fumarate, malate and aspartate via pyruvate carboxylase. Hence, the SCO2-/- cells are still able to maintain TCA activity via pyruvate carboxylase, which may be necessary for their survival. Moreover, we observed alterations in mTOR signaling between SCO2-deficient and -proficient cells. Strikingly, rpS6 and 4E-BP1 were dramatically less phosphorylated in SCO2-/- cells as compared to WT, which illustrated a shutdown of mTORC1 activity upon SCO2 loss. In turn, AKT and likely mTORC2 activity were higher in SCO2-/- as compared to WT cells as illustrated by increased GSK3 and AKT phosphorylation. This suggests a rewiring of SCO2-/- cells whereby high AKT activity is not paralleled by increased mTOR activity, and we speculate that this is likely due to activation of AMPK. We hypothesize AMPK is activated to maintain energy balance in the cells where mitochondrial ATP production is reduced due to the loss of complex IV function. The changes may be important to the survival and proliferation of SCO2-/- cells and may explain the observed metabolic rewiring due to the loss of the gene. Citation Format: Alexandre V. Zhdanov, Oro Uchenunu, Nadezda A. Brazhe, Evelina Nikelshparg, Dmitry E. Andreev, Ruslan I. Dmitriev, Olga V. Sosnovtseva, Ivan Topisirovic, Dmitri B. Papkovsky. Investigating the role of SCO2 in the metabolic adaptation of cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A20.

Degradation of key photosynthetic genes in the critically endangered semi-aquatic flowering plant Saniculiphyllum guangxiense (Saxifragaceae)

BackgroundPlastid gene loss and pseudogenization has been widely documented in parasitic and mycoheterotrophic plants, which have relaxed selective constraints on photosynthetic function. More enigmatic are sporadic reports of pseudogenization and loss of important photosynthesis genes in lineages thought to be fully photosynthetic. Here we report the complete plastid genome of Saniculiphyllum guangxiense, a critically endangered and phylogenetically isolated plant lineage, along with genomic evidence of reduced chloroplast function. We also report 22 additional plastid genomes representing the diversity of its containing clade Saxifragales, characterizing gene content and placing variation in a broader phylogenetic context.ResultsWe find that the plastid genome of Saniculiphyllum has experienced pseudogenization of five genes of the ndh complex (ndhA, ndhB, ndhD, ndhF, and ndhK), previously reported in flowering plants with an aquatic habit, as well as the surprising pseudogenization of two genes more central to photosynthesis (ccsA and cemA), contrasting with strong phylogenetic conservatism of plastid gene content in all other sampled Saxifragales. These genes participate in photooxidative protection, cytochrome synthesis, and carbon uptake. Nuclear paralogs exist for all seven plastid pseudogenes, yet these are also unlikely to be functional.ConclusionsSaniculiphyllum appears to represent the greatest degree of plastid gene loss observed to date in any fully photosynthetic lineage, perhaps related to its extreme habitat specialization, yet plastid genome length, structure, and substitution rate are within the variation previously reported for photosynthetic plants. These results highlight the increasingly appreciated dynamism of plastid genomes, otherwise highly conserved across a billion years of green plant evolution, in plants with highly specialized life history traits.

SLMP53-1 Inhibits Tumor Cell Growth through Regulation of Glucose Metabolism and Angiogenesis in a P53-Dependent Manner.

The Warburg effect is an emerging hallmark of cancer, which has the tumor suppressor p53 as its major regulator. Herein, we unveiled that p53 activation by (S)-tryptophanol-derived oxazoloisoindolinone (SLMP53-1) mediated the reprograming of glucose metabolism in cancer cells and xenograft human tumor tissue, interfering with angiogenesis and migration. Particularly, we showed that SLMP53-1 regulated glycolysis by downregulating glucose transporter 1 (GLUT1), hexokinase-2 (HK2), and phosphofructokinase-2 isoform 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) (key glycolytic enzymes), while upregulating the mitochondrial markers synthesis of cytochrome c oxidase 2 (SCO2), cytochrome c oxidase subunit 4 (COX4), and OXPHOS mitochondrial complexes. SLMP53-1 also downregulated the monocarboxylate transporter 4 (MCT4), causing the subsequent reduction of lactate export by cancer cells. Besides the acidification of the extracellular environment, SLMP53-1 further increased E-cadherin and reduced metalloproteinase-9 (MMP-9) expression levels in both cancer cells and xenograft human tumor tissue, which suggested the interference of SLMP53-1 in extracellular matrix remodeling and epithelial-to-mesenchymal transition. Consistently, SLMP53-1 depleted angiogenesis, decreasing endothelial cell tube formation and vascular endothelial growth factor (VEGF) expression levels. SLMP53-1 also exhibited synergistic growth inhibitory activity in combination with the metabolic modulator dichloroacetic acid. These data reinforce the promising application of the p53-activating agent SLMP53-1 in cancer therapy, by targeting p53-mediated pathways of growth and dissemination.

Metabolic transcriptional analysis on copper tolerance in moderate thermophilic bioleaching microorganism Acidithiobacillus caldus

Bioleaching, an alternative environmental smelting technology, typically uses high concentrations of heavy metal ions, especially in the subsequent phase, due to metal ion accumulation from the mineral. In this study, we analyzed the overall response of the bioleaching microorganism Acidithiobacillus caldus to copper stress through physiological and transcriptomic analyses. Scanning electron microscopy results showed higher extracellular polymeric substances secretion and cell aggregation under copper stress. Intracellular levels of glutamic acid, glycine and cysteine increased, favoring the synthesis of glutathione for maintenance of the oxidation-reduction state. GSH, during copper stress conditions, the activity of GSH-PX and CAT increased, resulting in reduced oxidative damage while maintaining stable intracellular pH. Higher unsaturated and cyclopropane fatty acid levels resulted in increased membrane fluidity and compactness and decreased ATP levels to support the energy requirements for stress resistance. Initially, H+-ATPase activity increased to provide energy for proton output and decreased later at higher copper ion stress. From transcriptome analysis, 140 genes were differentially expressed under low copper stress (1g/L), while 250 genes exhibited altered transcriptional levels at higher copper stress (3g/L). These differentially expressed genes were involved primarily in metabolic pathways such as energy metabolism, two-component systems, amino acid metabolism, and signal transduction. The Sox family cluster gene cluster involved in the conversion of thiosulfate to sulfate was upregulated in the sulfur metabolism pathway. In the oxidative phosphorylation pathway, genes participating in the synthesis of NADH oxidoreductase and cytochrome c oxidase, nuoL, cyoABD (cyoA, cyoB and cyoD) and cydAB (cydA and cydB), were downregulated. The TCS element ompR, closely associated with the osmotic pressure, exhibited active response, while Cu2+ efflux system gene cusRS was upregulated. In the amino acid metabolism, the glnA involved in nitrogen fixation was upregulated and promoted the synthesis of glutamine synthetase for reducing excessive oxidative stress. This study provides new insights into the mechanism underlying A. caldus response to heavy-metal ion stress under harsh bioleaching conditions.