Aconitase Research Articles

Light-Driven Carbon Fixation Using Photosynthetic Organelles in Artificial Photosynthetic Cells.

Building an artificial photosynthetic cell from scratchhelps to understandthe working mechanismsof chloroplasts.It is a challenge to achieve carbon fixation triggered by photosynthetic organelles in an artificial cell. ATP synthase and photosystem II (PSII)are purified and reconstituted onto the phospholipid membrane to fabricate photosynthetic organelles. With the integration of phycocyanin, the ATP production yield increases by 2.51-fold due to the enhanced light harvesting capability. The carbon fixation pathway is established by converting α-oxoglutarate to acetyl-CoA and oxaloacetate with cascade enzyme reactions including the isocitratedehydrogenase (IDH), aconitase (ACO), and ATP citrate lyase (ACL).The photosynthetic organelles,phycocyanin,and carbon fixation pathway are encapsulatedinto giant unilamellar vesiclesto obtain artificial photosynthetic cells, which convert α-oxoglutarate to acetyl-CoA and oxaloacetate inside artificial cells upon light irradiation. The acetyl-CoA and oxaloacetatearethe most important intermediate products in the cellularmetabolic networks for the synthesis of cholesterol and fatty acids. Our results provide a way for efficient light energy conversion to produce ATP and fix CO2, and pave the path to buildautonomous artificial cells with more complicated metabolic networks.

Divergence in MiRNA targeting of AchAco and its role in citrate accumulation in kiwifruit

BackgroundMicroRNA (miRNA) is a crucial post-transcriptional regulatory factor in plant growth and development. Duplicated genes often exhibit functional divergence due to competition for the identical miRNA binding sites. Kiwifruit (Actinidia spp.) is an economically significant horticultural crop renowned for its distinctive flavor, which is largely attributable to elevated citrate levels during fruit development. However, the mechanisms through which miRNA-targeted modules evolve following duplication events and regulate citrate biosynthesis, thereby influencing the unique flavor profile of kiwifruits, remain poorly understood.ResultsIn this study, we examined the expression patterns of miRNAs and interactions with their targets in kiwifruit fruit samples from various pulp tissues and developmental stages. Our analysis identified 46 miRNAs, comprising 44 known miRNAs and two novel/kiwifruit-specific miRNAs, which targeted a total of 1,474 genes. Correlation analysis revealed weak relationships between the expression levels of miRNAs and their target genes. Among these targets, 27 tandemly duplicated genes, and 782 whole genome duplication (WGD) genes exhibited a loss of miRNA binding sites in one of their duplicated copies. Furthermore, weighted gene co-expression network analysis demonstrated that most duplicated genes clustered into distinct gene modules. These findings suggest that the loss of miRNA targets following duplications contributes to expression divergence among gene duplicates, thereby facilitating stable gene expression within the miRNA-targeted network. For instance, the aconitate hydratase genes AchAco4 and AchAco6 were each targeted by different miRNAs, ach-miR-3774 and ach-miR-10371, respectively. Notably, these genes exhibited distinct expression patterns compared to their duplicated counterparts.ConclusionsThis study enhances our understanding of how the miRNA-AchAco module regulates citrate content and provides insights into the molecular network that influences the flavor profile of kiwifruit.Clinical trial numberNot applicable.

The role of peritumoral electroacupuncture in regulating cuproptosis for sensitization of chemotherapy efficacy in mice with triple-negative breast cancer

To explore the enhanced sensitization effect of peritumoral electroacupuncture (PEA) on doxorubicin (DOX) chemotherapy in mice with triple-negative breast cancer (TNBC). Eighteen female Balb/c mice were randomly divided into the model, DOX, and EA+DOX groups, with 6 mice in each group. TNBC cells were injected into the mammary fat pad of mice to establish the breast cancer-bearing mice model. Mice of the DOX and EA+DOX groups were injected intraperitoneally with DOX solution at 4 mg/kg once a week for 4 weeks. For mice of the EA+DOX group, 4 filiform needles were inserted surrounding tumor tissues, followed by giving the mice with EA (1 mA, 2 Hz/100 Hz) stimulation for 3 min, once a week for 4 weeks. During the intervention, the tumor volume was measured every two days, and at the end of intervention, the weight of the tumor tissue was measured. The expression of Ki67 and proliferating cell nuclear antigen (PCNA) in tumor tissues was detected by immunohistochemistry. The protein expression levels of iron redox protein 1 (FDX1), lipoic acid synthase (LIAS), aconitase 2 (ACO2) and NADH：ubiquinone oxidoreductase core subunit V1 (NDUFV1), dihydrolipoamide S-acetyltransferase(DLAT), dihydrolipoyl succinyltransferase(DLST) in tumor tissues were detected by Western blot. The content of copper ions, pyruvic acid and α-ketoglutaric acid, succinic acid in tumor tissues was detected by copper ion kit, and the content of reactive oxygen species (ROS) in tumor tissues of mice was detected by fluorescence method. Compared with the model group, the tumor volume and weight, the expression levels of Ki67 and PCNA in tumor tissues were decreased (P<0.05, P<0.01) in the DOX and EA+DOX groups. The improvement of the above indicators was more significant in the EA+DOX group than that in the DOX group (P<0.05, P<0.01). Compared with the model and DOX groups, the contents of ROS, copper ions, pyruvic acid and α-ketoglutaric acid were increased (P<0.05, P<0.01), while the contents of succinic acid, and the expressions of FDX1, LIAS, ACO2 and NDUFV1, DLAT, DLST proteins were decreased (P<0.01) in the EA+DOX group. The combination of PEA and DOX can effectively control the growth rate of tumors in mice with TNBC, which may contribute to its function in enhancing the chemotherapy efficacy of DOX on TNBC by promoting cuproptosis.

Omics-centric evidences of fipronil biodegradation by Rhodococcus sp. FIP_B3

The widespread use of the pesticide fipronil in domestic and agriculture sectors has resulted in its accumulation across the environment. Its use to assure food security has inadvertently affected soil microbiome composition, fertility and, ultimately, human health. Degradation of residual fipronil present in the environment using specific microbial species is a promising strategy for its removal. The present study delves into the omics approach for fipronil biodegradation using the native bacterium Rhodococcus sp. FIP_B3. It has been observed that within 40 days, nearly 84% of the insecticide gets degraded. The biodegradation follows a pseudo-first-order kinetics (k = 0.0197/d with a half-life of ∼11 days). Whole genome analysis revealed Cytochrome P450 monooxygenase, peroxidase-related enzyme, haloalkane dehalogenase, 2-nitropropane dioxygenase, and aconitate hydratase are involved in the degradation process. Fipronil-sulfone, 5-amino-1-(2-chloro-4-(trifluoromethyl)phenyl)-4- ((trifluoromethyl)sulfonyl)-1H-pyrazole-3-carbonitrile, (E)-5-chloro-2-oxo-3- (trifluoromethyl)pent-4-enoic acid, 4,4,4-trifluoro-2-oxobutanoic acid, and 3,3,3- trifluoropropanoic acid were identified as the major metabolites that support the bacterial degradation of fipronil. In-silico molecular docking and molecular dynamic simulation-based analyses of degradation pathway intermediates with their respective enzymes have indicated stable interactions with significant binding energies (−5.9 to −9.7 kcal/mol). These results have provided the mechanistic cause of the elevated potential of Rhodococcus sp. FIP_B3 for fipronil degradation and will be advantageous in framing appropriate strategies for the bioremediation of fipronil-contaminated environment.

Transcriptome analyses reveal molecular mechanisms of novel compound heterozygous ACO2 variants causing infantile cerebellar retinal degeneration.

Infantile cerebellar retinal degeneration (ICRD) (OMIM #614559) is a rare autosomal recessive inherited disease associated with mutations in the aconitase 2 (ACO2) gene. We report a Chinese girl with novel compound heterozygous variants in ACO2, who presented at 7 months of age with psychomotor retardation, truncal hypotonia, and ophthalmologic abnormalities. This study aims to investigate the potential molecular mechanisms underlying ACO2 deficiency-induced neuropathy. Whole exome sequencing was performed on family members to screen for potential pathogenic mutations, followed by Sanger sequencing for validation. Mitochondrial aconitase activity and mitochondrial DNA (mtDNA) copy number were measured using an aconitase activity detection kit and quantitative PCR, respectively. Transcriptome expression profiles from patient cells, and cerebellar and retinal organoids retrieved from the GEO database were integrated. Functional enrichment analysis and protein-protein interaction networks were used to identify key molecules, and their expression levels were validated using Western blot analysis. Genetic testing revealed novel compound heterozygous variations in the proband's ACO2 gene (NM:001098), including c.854A>G (p.Asn285Ser) and c.1183C>T (p.Arg395Cys). Predictive analysis of the tertiary structure of the ACO2 protein suggests that both p.Asn285Ser and p.Arg395Cys affect the binding ability of ACO2 to ligands. The mitochondrial aconitase activity and mtDNA copy number in the proband's leukocytes were significantly reduced. Transcriptomic data analysis identified 80 key candidate genes involved in ACO2-related neuropathy. Among these, LRP8 and ANK3, whose gene expression levels were significantly positively correlated with ACO2, were further validated by Western blot analysis. This study expands the spectrum of pathogenic ACO2 variants, elucidates the potential molecular mechanisms underlying ACO2-related neuropathy, provides in-depth support for the pathogenicity of ACO2 genetic variations, and offers new insights into the pathogenesis of ICRD.

The Key Enzymes of Carbon Metabolism and the Glutathione Antioxidant System Protect Yarrowia lipolytica Yeast Against pH-Induced Stress.

In this study, we first thoroughly assayed the response of the key enzymes of energy metabolism and the antioxidant system in Yarrowia lipolytica yeast at extreme pH. The activity of the tricarboxylic acid cycle enzymes, namely NAD-dependent isocitrate dehydrogenase, aconitate hydratase, NAD-dependent malate dehydrogenase, and fumarate hydratase, NADPH-producing enzymes of glucose-6-P dehydrogenase and NADP-dependent isocitrate dehydrogenase, and the enzymes of the glutathione system was assessed. All the enzymes that were tested showed a significant induction contrary to some decrease in the aconitate hydratase activity with acidic and alkaline stress. It is probable that a change in the enzyme activity in the mitochondria matrix is involved in the regulation of the cellular metabolism of Y. lipolytica, which allows the species to prosper at an extreme ambient pH. It distinguishes it from any other type of ascomycete. A close relationship between the induction of the Krebs cycle enzymes and the key enzymes of the glutathione system accompanied by an increased level of reduced glutathione was shown. The assumption that the increased activity of the Krebs cycle dehydrogenases and promotion of the pentose phosphate pathway at pH stress launches a set of events determining the adaptive response of Y. lipolytica yeast.

S-nitrosylation of aconitase-2 facilitates coronary microembolization-induced cardiac injury by suppressing iron-sulfur cluster assembly

Abstract Background Coronary microembolization (CME) is a common reason for periprocedural myocardial infarction after coronary interventions. S-nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in the pathogenesis of several cardiovascular diseases. Purpose To explore the role of SNO of aconitase-2 (ACO2) in CME-induced cardiac injury, as well as the mechanism by which SNO-ACO2 modulates myocardial injury in response to CME. Methods CME models were established in C57BL/6J mice by injecting 300,000 polyethylene microspheres (diameter 9 µm) into the left ventricle chamber with the occlusion of the ascending aorta. Cardiac function was evaluated by echocardiography. Histological and serum analysis was performed to evaluate myocardial injury. Myocardial samples were scanned for S-nitrosylated proteins using biotin-switch procedures and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. SNO sites of ACO2 were further identified through LC-MS/MS. De-nitrosylation of ACO2 was achieved by the mutation of SNO site or overexpression of the de-nitrosylation enzyme thioredoxin 1 (TRX1). Interacting effectors of SNO- ACO2 were screened through LC-MS/MS and confirmed by coimmunoprecipitation. Neonatal rat ventricular myocytes (NRVMs) were used for in vitro study. The cellular ACO2 activity was analysed using an ACO2 enzyme activity assay kit. Mitochondrial morphology and function were assessed by fluorescent staining and mitochondrial stress testing. Results Cardiac injury was exacerbated by CME as demonstrated by impaired cardiac contractile function, morphological changes, and increased fibrosis, microinfarct size and serum troponin I level. We identified 789 S-nitrosylated proteins in CME mouse hearts, and ACO2 is one of the highly S-nitrosylated proteins. The increased level of SNO-ACO2 was verified by immunoblot assay in CME mouse hearts and hypoxia-stimulated NRVMs. SNO site of ACO2 at cysteine 126 was identified by LC-MS/MS and confirmed in NRVMs. De-nitrosylation of ACO2 by the mutation of cysteine 126 or overexpression of TRX1 both greatly improved the activity of ACO2 by 39.7% and 31.4% respectively, furthermore restored the mitochondrial function, and attenuated CME-induced cardiac injury. Mechanistically, SNO-ACO2 at cysteine 126 suppressed the interaction between ACO2 and NFU1, an iron-sulfur (Fe-S) cluster scaffold protein. The dissociation from NFU1 deprived ACO2 of the Fe-S cluster and the enzyme activity, thereby promoting the disruption of the mitochondrial tricarboxylic acid cycle and cardiac injury. Conclusions Our data defined a previously unrecognized role of SNO-ACO2 in CME-induced cardiac injury. We demonstrated that SNO-ACO2 at cysteine 126 disrupted ACO2/NFU1 interaction to exacerbate myocardial injury after CME. Our results suggested that de-nitrosylation of ACO2 may provide a new therapeutic target for the treatment of CME.

Hepatotoxic doses of copper sulfate induce metabolic memory in the redox system, which has an age-dependent nature

Abstract. We tested the hypothesis that ageing is a consequence of the formation of metabolic memory and the possible role of the redox system as a basic, evolutionarily ancient system of metabolism regulation in stable metabolic patterns formation or metabolic states chronisation changed during adaptation. Three sequential intraperitoneal administration of copper sulfate to young (3-month-old Wistar rats) and old animals (20 months) at a dose of 1 mg/100 g of body weight (33% of the lethal dose) were used as adaptive inducers of the redox system. The amount of lipid hydroperoxides in mitochondria, cytosol in liver cells and blood serum, the activity of mitochondrial aconitate hydratase as an indicator of oxidative stress and the activity of a number of antioxidant enzymes were determined to assess the initial metabolic states, i. e. before exposure and 1, 30 and 60 days after exposure to copper sulfate on the body. It was shown that the amount of lipid hydroperoxides (LOOHs) in the liver mitochondria and blood serum of old rats before exposure to copper sulfate was more than 30% lower than in young animals, while the aconitase activity (an indicator of oxidative stress) was the same in animals of these ages. A lower amount of LOOHs coincided with an increased glutathione peroxidase activity in old animals. In old rats, the increased amount of LOOHs induced by copper ions was preserved even after eliminating copper from the body 30 days after administration. At the same time, it was restored to the original level in the young animals. The glutathione peroxidase and aconitase activity in mitochondria remained below the control values even after the inducer elimination, and this was age-depending. The obtained results do not contradict the hypothesis of metabolic memory's role in ageing mechanisms. We postulate a relationship between the duration of maintenance of altered metabolic patterns and the polyfunctionality of enzymes and other metabolites. _________________________________________________________________________________________ Keywords: ageing; redox system; metabolic memory; lipid hydroperoxides; antioxidant enzymes; chronic states.

Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

The Secretome of Adult Murine Hookworms Is Shaped by Host Expression of STAT6.

Co-evolutionary adaptation of hookworms with their mammalian hosts has been selected for immunoregulatory excretory/secretory (E/S) products. However, it is not known whether, or if so, how host immunological status impacts the secreted profile of hematophagous adult worms. This study interrogated the impact of host Signal transducer and activator of transcription 6 (STAT6) expression during the experimental evolution of hookworms through the sequential passage of the life cycle in either STAT6 deficient or WT C57BL/6 mice. Proteomic analysis of E/S products by LC-MS showed increased abundance of 15 proteins, including myosin-3, related to muscle function, and aconitate hydratase, related to iron homeostasis. However, most E/S proteins (174 of 337 unique identities) were decreased, including those in the Ancylostoma-secreted protein (ASP) category, and metallopeptidases. Several identified proteins are established immune-modulators such as fatty acid-binding protein homologue, cystatin, and acetylcholinesterase. Enrichment analysis of InterPro functional categories showed down-regulation of Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP), Astacin-like metallopeptidase, Glycoside hydrolase, and Transthyretin-like protein groups in STAT6 KO-adapted worms. Taken together, these data indicate that in an environment lacking Type 2 immunity, hookworms alter their secretome by reducing immune evasion proteins- and increasing locomotor- and feeding-associated proteins.

Concept of Normativity in Multi-Omics Analysis of Axon Regeneration.

Transcriptomes and proteomes can be normalized with a handful of RNAs or proteins (or their peptides), such as GAPDH, β-actin, RPBMS, and/or GAP43. Even with hundreds of standards, normalization cannot be achieved across different molecular mass ranges for small molecules, such as lipids and metabolites, due to the non-linearity of mass by charge ratio for even the smallest part of the spectrum. We define the amount (or range of amounts) of metabolites and/or lipids per a defined amount of a protein, consistently identified in all samples of a multiple-model organism comparison, as the normative level of that metabolite or lipid. The defined protein amount (or range) is a normalized value for one cohort of complete samples for which intrasample relative protein quantification is available. For example, the amount of citrate (a metabolite) per µg of aconitate hydratase (normalized protein amount) identified in the proteome is the normative level of citrate with aconitase. We define normativity as the amount of metabolites (or amount range) detected when compared to normalized protein levels. We use axon regeneration as an example to illustrate the need for advanced approaches to the normalization of proteins. Comparison across different pharmacologically induced axon regeneration mouse models entails the comparison of axon regeneration, studied at different time points in several models designed using different agents. For the normalization of the proteins across different pharmacologically induced models, we perform peptide doping (fixed amounts of known peptides) in each sample to normalize the proteome across the samples. We develop Regen V peptides, divided into Regen III (SEB, LLO, CFP) and II (HH4B, A1315), for pre- and post-extraction comparisons, performed with the addition of defined, digested peptides (bovine serum albumin tryptic digest) for protein abundance normalization beyond commercial labeled relative quantification (for example, 18-plex tandem mass tags). We also illustrate the concept of normativity by using this normalization technique on regenerative metabolome/lipidome profiles. As normalized protein amounts are different in different biological states (control versus axon regeneration), normative metabolite or lipid amounts are expected to be different for specific biological states. These concepts and standardization approaches are important for the integration of different datasets across different models of axon regeneration.

The tricarboxylic acid cycle is inhibited under acute stress from carbonate alkalinity in the gills of Eriocheir sinensis

Owing to population growth and environmental pollution, freshwater aquaculture has been rapidly shrinking in recent years. Aquaculture in saline-alkaline waters is a crucial strategy to meet the increasing demand for aquatic products. The Chinese mitten crab is an important economic food in China, but the molecular mechanism by which it tolerates carbonate alkalinity (CA) in water remains unclear. Here, we found that enzyme activities of the tricarboxylic acid (TCA) cycle in the gills, such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase, were markedly reduced under CA stress induced by 40 mM NaHCO3. Secondly, the TCA cycle in the gills is inhibited under acute CA stress, according to proteomic and metabolomic analyses. The expressions of six enzymes, namely aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, dihydrolipoyl dehydrogenase, succinate-CoA ligase, and malate dehydrogenase, were downregulated, resulting in the accumulation of phosphoenolpyruvic acid, citric acid, cis-aconitate, and α-ketoglutaric acid. Finally, we testified that if the TCA cycle is disturbed by malonate, the survival rate increases in CA water. To our knowledge, this is the first study to show that the TCA cycle in the gills is inhibited under CA stress. Overall, the results provide new insights into the molecular mechanism of tolerance to saline-alkaline water in crabs, which helped us expand the area for freshwater aquaculture and comprehensively understand the physiological characteristics of crab migration.

Potato glycoside alkaloids exhibit antifungal activity by regulating the tricarboxylic acid cycle pathway of Fusarium solani.

Fusarium solani is a pathogenic fungus that causes significant harm, leading to crop yield reduction, fruit quality reduction, postharvest decay, and other diseases. This study used potato glycoside alkaloids (PGA) as inhibitors to investigate their effects on the mitochondrial structure and tricarboxylic acid (TCA) cycle pathway of F. solani. The results showed that PGA could inhibit the colony growth of F. solani (54.49%), resulting in the disappearance of the mitochondrial membrane and the loss of contents. PGA significantly decreased the activities of aconitase (ACO), isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH), fumarase (FH), malate dehydrogenase (MDH), succinyl-CoA synthetase (SCS), and increased the activity of citrate synthase (CS) in F. solani. After PGA treatment, the contents of acetyl coenzyme A (CoA), citric acid (CA), malic acid (L-MA), and α-ketoglutaric acid (α-KG) in F. solani were significantly decreased. The contents of isocitric acid (ICA), succinyl coenzyme A (S-CoA), succinic acid (SA), fumaric acid (FA), and oxaloacetic acid (OA) were significantly increased. Transcriptomic analysis showed that PGA could significantly affect the expression levels of 19 genes related to TCA cycle in F. solani. RT-qPCR results showed that the expression levels of ACO, IDH, α-KGDH, and MDH-related genes were significantly down-regulated, and the expression levels of SDH and FH-related genes were significantly up-regulated, which was consistent with the results of transcriptomics. In summary, PGA can achieve antifungal effects by reducing the tricarboxylic acid cycle's flow and regulating key genes' expression levels. This study reveals the antifungal mechanism of PGA from the perspective of TCA cycle, and provides a theoretical basis for the development and application of PGA as a biopesticide.

Surface Proteome of Extracellular Vesicles and Correlation Analysis Reveal Breast Cancer Biomarkers

Breast cancer (BC) is the second most frequently diagnosed cancer and accounts for approximately 25% of new cancer cases in Canadian women. Using biomarkers as a less-invasive BC diagnostic method is currently under investigation but is not ready for practical application in clinical settings. During the last decade, extracellular vesicles (EVs) have emerged as a promising source of biomarkers because they contain cancer-derived proteins, RNAs, and metabolites. In this study, EV proteins from small EVs (sEVs) and medium EVs (mEVs) were isolated from BC MDA-MB-231 and MCF7 and non-cancerous breast epithelial MCF10A cell lines and then analyzed by two approaches: global proteomic analysis and enrichment of EV surface proteins by Sulfo-NHS-SS-Biotin labeling. From the first approach, proteomic profiling identified 2459 proteins, which were subjected to comparative analysis and correlation network analysis. Twelve potential biomarker proteins were identified based on cell line-specific expression and filtered by their predicted co-localization with known EV marker proteins, CD63, CD9, and CD81. This approach resulted in the identification of 11 proteins, four of which were further investigated by Western blot analysis. The presence of transmembrane serine protease matriptase (ST14), claudin-3 (CLDN3), and integrin alpha-7 (ITGA7) in each cell line was validated by Western blot, revealing that ST14 and CLDN3 may be further explored as potential EV biomarkers for BC. The surface labeling approach enriched proteins that were not identified using the first approach. Ten potential BC biomarkers (Glutathione S-transferase P1 (GSTP1), Elongation factor 2 (EEF2), DEAD/H box RNA helicase (DDX10), progesterone receptor (PGR), Ras-related C3 botulinum toxin substrate 2 (RAC2), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), Aconitase 2 (ACO2), UTP20 small subunit processome component (UTP20), NEDD4 binding protein 2 (N4BP2), Programmed cell death 6 (PDCD6)) were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. In total, 846 surface proteins were identified from the second approach, of which 11 were already known as BC markers. This study supports the proposition that Evs are a rich source of known and novel biomarkers that may be used for non-invasive detection of BC. Furthermore, the presented datasets could be further explored for the identification of potential biomarkers in BC.

Metabolic engineering of Shewanella oneidensis to produce glutamate and itaconic acid

Shewanella oneidensis is a gram-negative bacterium known for its unique respiratory capabilities, which allow it to utilize a wide range of electron acceptors, including solid substrates such as electrodes. For a future combination of chemical production and electro-fermentation, the goal of this study was to expand its product spectrum. S. oneidensis was metabolically engineered to optimize its glutamate production and to enable production of itaconic acid. By deleting the glutamate importer gltS for a reduced glutamate uptake and pckA/ptA to redirect the carbon flux towards the TCA cycle, a ∆3 mutant was created. In combination with the plasmid pG2 carrying the glutamate dehydrogenase gdhA and a specific glutamate exporter NCgl1221 A111V, a 72-fold increase in glutamate concentration compared to the wild type was achieved. Along with overexpression of gdhA and NCgl1221 A111V, the deletion of gltS and pckA/ptA as well as the deletion of all three genes (∆3) was examined for their impact on growth and lactate consumption. This showed that the redirection of the carbon flux towards the TCA cycle is possible. Furthermore, we were able to produce itaconic acid for the first time with a S. oneidensis strain. A titer of 7 mM was achieved after 48 h. This suggests that genetic optimization with an expression vector carrying a cis-aconitate decarboxylase (cadA) and a aconitate hydratase (acnB) along with the proven redirection of the carbon flux to the TCA cycle enabled the production of itaconic acid, a valuable platform chemical used in the production of a variety of products.Key points•Heterologous expression of gdhA and NCgl1221_A111V leads to higher glutamate production.•Deletion of ackA/pta redirects carbon flux towards TCA cycle.•Heterologous expression of cadA and acnB enables itaconic acid production.

Liquid semen storage-induced alteration in the protein composition of turkey (Meleagris gallopavo) spermatozoa

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

SkQ1 improves immune status and normalises the activity of antioxidant and nadph-generating enzymes in adjuvant-induced rheumatoid arthritis in rats

Rheumatoid arthritis (RA) is a severe systemic autoimmune inflammatory disease. Oxidative stress and excessive formation of mitochondrial reactive oxygen species (ROS) are now considered to be the central pathogenetic mechanisms of connective tissue component destruction and the factors responsible for a highly active inflammatory process and autoimmune response. The aim of the present work was to evaluate the effect of mitochondrial-directed antioxidant 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on immune status, intensity of free radical-induced oxidation and functioning of antioxidant system (AOS) and NADPH-generating enzymes in rats with adjuvant-induced RA. Laboratory animals were divided into 4 groups: a control group; a group of animals with RA; animals injected intraperitoneally with SkQ1 at a dose of 1250 nmol/kg every 24 hours during the next 8 days from day 7 of RA development, and animals given SkQ1 at a dose of 625 nmol/kg according to the above scheme. Material for the study was taken on day 15 after the start of the experiment. The rate of erythrocyte sedimentation, the level of circulating immune complexes, and the concentration of immunoglobulins A, M and G were determined in rats by enzyme immunoassay. The intensity of free radical-induced oxidation was assessed by iron-induced biochemiluminescence, diene conjugate content and aconitate hydratase activity. Enzyme activity and metabolite content in animal tissues were analysed spectrophotometrically. The results of the work showed that the development of RA was associated with an increase in the indicators of immune response and intensity of free radical-induced oxidation. The development of an imbalance in AOS functioning and an increase in the activity of NADPH-generating enzymes was also observed. The administration of SkQ1 resulted in a dose-dependent change in the oxidative status indicators towards the control, which was accompanied by the normalization of the immune status parameters. It seems that the tested compound decreased the level of mitochondrial ROS, resulting in an inhibition of the inflammatory response. The consequence of these changes could be inhibition of free radical generation by immunocompetent cells and subsequent mitigation of the severity of oxidative stress in the tissues.

ACO2 deficiency increases vulnerability to Parkinson’s disease via dysregulating mitochondrial function and histone acetylation-mediated transcription of autophagy genes

Parkinson’s disease (PD) is characterized by α-synuclein aggregation in dopaminergic (DA) neurons, which are sensitive to oxidative stress. Mitochondria aconitase 2 (ACO2) is an essential enzyme in the tricarboxylic acid cycle that orchestrates mitochondrial and autophagic functions to energy metabolism. Though widely linked to diseases, its relation to PD has not been fully clarified. Here we revealed that the peripheral ACO2 activity was significantly decreased in PD patients and associated with their onset age and disease durations. The knock-in mouse and Drosophila models with the A252T variant displayed aggravated motor deficits and DA neuron degeneration after 6-OHDA and rotenone-induction, and the ACO2 knockdown or blockade cells showed features of mitochondrial and autophagic dysfunction. Moreover, the transcription of autophagy-related genes LC3 and Atg5 was significantly downregulated via inhibited histone acetylation at the H3K9 and H4K5 sites. These data provided multi-dimensional evidences supporting the essential roles of ACO2, and as a potential early biomarker to be used in clinical trials for assessing the effects of antioxidants in PD. Moreover, ameliorating energy metabolism by targeting ACO2 could be considered as a potential therapeutic strategy for PD and other neurodegenerative disorders.

PTP4A1 promotes oral squamous cell carcinoma (OSCC) metastasis through altered mitochondrial metabolic reprogramming

PTP4A1 (Protein tyrosine phosphatase 4A1) is a protein tyrosine phosphatase that regulates a range of pro-oncogenic signaling pathways. Here, we report a novel role for PTP4A1 in oral squamous cell carcinoma (OSCC) growth and development. We show that PTP4A1 is frequently overexpressed in OSCC cells and tissues compared to adjacent non-tumor tissue. In OSCC, the overexpression of PTP4A1 increased cell growth and invasion in vitro, and enhanced tumor progression in vivo. At the molecular level, PTP4A1 was found to regulate mitochondrial metabolic reprogramming to enhance the invasive capacity of OSCC cells. Mechanistically, these effects were mediated through binding to pyruvate kinase isoenzyme M2 (PKM2) to promote its expression and aconitase 2 (ACO2) to enhance its degradation. Together, these data reveal PTP4A1 as a viable target for OSCC therapeutics.

Iterative optimization of exogenous genes and redirection of metabolic flux for enhanced itaconate biosynthesis in engineered Escherichia coli

BackgroundItaconate or itaconic acid (IA), a sustainable and valuable platform chemical, has garnered significant attention due to its potential as an alternative petroleum-derived compound. In the past, Aspergillus terreus offered a high titer of IA, but it still faced the challenges of low productivity. To overcome the problem, a fast-growing Escherichia coli BW25113 was involved consolidating the itaconate production pathway, eliminating byproduct-forming pathways, and enhancing intermediate levels to facilitate large-scale production. MethodE. coli was engineered through the deletion of isocitrate decarboxylase (Icd) and the overexpression of pyruvate carboxylase (CgPyc), cis-aconitate decarboxylase (AtCadA) and aconitate hydratase (ENAcnA) to facilitate the IA biosynthesis pathway. A B0015 terminator was encoded behind AtCadA to improve transcription efficiency as well as poxB gene was deleted to accelerate carbon influx towards the tricarboxylic acid cycle instead of forming the byproduct acetate. A semi-continuous batch fermentation approach was employed for large-scale IA biosynthesis to address ATP insufficiency in long-termed fermentation. Significant findingThe engineered strain AB15C/Δicd improved IA titer by 20 % compared to the control strain. By deleting the acetate forming pathway under acidic condition, AB15C/ΔicdΔpoxB increased itaconate production. Overexpression of ENAcnA in AB15CEN*/ΔicdΔpoxB strain resulted in 13.8 g/L of IA. Finally, a semi-continuous batch fermentation was implemented, leading to an IA accumulation of 35.5 g/L after 4 batches.