succinyl-CoA Synthetase Research Articles

Exploring microbial players for metagenomic profiling of carbon cycling bacteria in sundarban mangrove soils

The Sundarbans, the world’s largest tidal mangrove forest, acts as a crucial ecosystem for production, conservation, and the cycling of carbon and nitrogen. The study explored the hypothesis that microbial communities in mangrove ecosystems exhibit unique taxonomic and functional traits that play a vital part in carbon cycling and ecosystem resilience. Using metagenomic analysis to evaluate microbial communities in mangrove and non-mangrove environment, evaluating their composition, functional functions, and ecological relevance. The analysis revealed distinct microbial profiles, in mangrove and non-mangrove environments, with bacteria, proteobacteria, and viruses being the most prevalent groups, with varying abundances in each environment. Functional and taxonomical analysis identified genes involved in carbon regulation, including Triacylglycerol lipase, NarG, DsrB, DNA-binding transcriptional dual regulator CRP, Vanillate O-demethylase oxygenase, succinate-CoA ligase, Tetrahydrofolate ligase, Carboxylase, Ribulose-1,5-bisphosphate carboxylase/oxygenase, Glycine hydroxymethyltransferase, MAG: urease, Endosymbiont of Oligobrachia haakonmosbiensis, Ribulose bisphosphate carboxylase, Aconitate hydratase AcnA, and nitrous oxide reductase, suggesting the metabolic versatility of these microbial communities for carbon cycling. The findings emphasize the key role of microbial activity in preserving mangrove ecosystem health and resilience, highlighting the intricate interplay between microbial diversity, functional capabilities, and environmental factors.

Fluxomics Combined with Shotgun Proteomics Reveals a Differential Response of Bovine Kidney Cells to Extracellular Palmitic and α-Linolenic Acid.

Pyruvate carboxylase (PC) catalyzes the formation of oxaloacetate, a TCA cycle intermediate and gluconeogenic substrate. Altering saturated to unsaturated fatty acid ratio alters PC expression, suggesting a central role in mediating carbon flow through metabolic pathways. Herein, we describe changes in metabolic flux of TCA cycle intermediates and proteome in Madin Darby bovine kidney (MDBK) cells with PC expression knocked-down (PC-KD), overexpressed (PC-OE), unaltered using a Scramble control, or cells pretreated for 21 h with vehicle control bovine serum albumin (BSA) or different ratios of palmitic acid (P) and α-linolenic acid (L) ranging from 1 mM P:0 mM L (1P:0L) to 0P:1L. All cells were collected for proteome analysis and to measure [U-13C] pyruvate flux or oxidation of [1-14C] palmitic acid and [U-14C] lactate. Compared to Scramble, 13C enrichment of all TCA cycle intermediates was greater in PC-OE, but all were reduced in PC-KD except succinate. Proteins greater in abundance in both cell lines included solute transporters, propionyl CoA carboxylase, and fatty acid binding protein 3. Relative to BSA, 1P:0L increased cell death and increased 13C flux to citrate but decreased enrichment of succinate. Abundance of citrate synthase, aconitase, glutamine aminotransferases, and succinyl CoA synthetases was greater in 1P:0L, but not different in other pretreatments. Results indicate preferential utilization of pyruvate and amino acids by 1P:0L cells whereas 0P:1L treated cells show preference for α-linolenic acid metabolism. PC regulates metabolic flux, C18:3n-3 cis prevents lipotoxicity, and both alterations in PC and addition of C18:3n-3 cis promote oxidation of fatty acids.

Exploitation of Key Regulatory Modules and Genes for High-Salt Adaptation in Schizothoracine by Weighted Gene Co-Expression Network Analysis

Schizothoracine fishes in saltwater lakes of the Tibetan Plateau are important models for studying the evolution and uplift of the Tibetan Plateau. Examining their adaptation to the high-salt environment is interesting. In this study, we first assembled the RNA-Seq data of each tissue of G. przewalskii, G. selincuoensis, and G. namensis from Qinghai Lake, Selincuo Lake, and Namtso Lake, respectively, obtained by the group previously. After obtaining reliable results, the adaptation of the gills, kidneys, and livers of the three species to the high-salinity environment was assessed by weighted gene co-expression network analysis (WGCNA). Using module eigengenes (ME), 21, 22, and 22 gene modules were identified for G. przewalskii, G. selincuoensis, and G. nemesis, respectively. Functional clustering analysis of genes in the significant association module identified several genes associated with osmolarity-regulated potential KEGG pathways in the gills of three species of Schizothoracine fish. Th17 cell differentiation pathway was up-regulated in the gills of all three species; histocompatibility class 2 II antigen and E alpha (h2-ea) were up-regulated genes in this pathway. Functional clustering analysis of genes in apparently related modules in the kidney unveiled several differential KEGG pathways. The pentose phosphate pathway was up-regulated in the three Schizothoracine fishes, and glucose-6-phosphate dehydrogenase (g6pd) was an up-regulated gene in this pathway. In the livers of the three Schizothorax species, the propanoate metabolism pathway was up-regulated, and succinate-CoA ligase GDP-forming subunit beta (suclg2) was an up-regulated gene in this pathway. The above analyses provide reference data for the adaptation of Schizothorax to high-salt environments and lay the foundation for future studies on the adaptive mechanism of Schizothorax in the plateau. These results partly fill the void in the knowledge gap in the survival adaptations of Schizothoracine fishes to highland saline lakes.

Impact of missense mutations on the structure–function relationship of human succinyl-CoA synthetase using in silico analysis

The encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with succinyl-CoA synthetase (SCS) deficiency caused by pathogenic variants in genes encoding its two subunits. SCS is a mitochondrial enzyme involved in several metabolic pathways and acts as a heterodimer composed of α and β subunits encoded by SUCLG1 and SUCLA2 genes, respectively. The purpose of this study was to analyze the effects of the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) by applying, using different prediction tools, a filtering strategy, on the 343 and 365 nsSNPs found in SUCLG1 and SUCLA2 genes, respectively, retrieved from the databases, then to evaluate their structural and functional effects using homology modeling and molecular docking. Results showed that most deleterious mutations selected for structural analysis were located in loop regions critical for protein stability and function, especially, variants altering glycine and proline residues in these regions supporting their importance. We also showed that variants leading to hydrophobic and hydrophilic residues can destabilize the folding and binding of the protein. Molecular docking has also been used to identify the most important regions of ligand binding site (CoA binding site, ADP-Mg2+ binding site and phosphate ion binding site) and between the two subunits themselves, which mainly involving the ligase CoA domain. Our structural analysis, performed on selected nsSNP, are in accordance with experimental studies reported in the literature and predicted that they would responsible to either nonfunctional protein, subunit instability resulting in reduced amounts of misassembled protein, or in a protein unable to phosphorylate ADP.

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Ketogenic β-hydroxybutyrate regulates β-hydroxybutyrylation of TCA cycle-associated enzymes and attenuates disease-associated pathologies in Alzheimer's mice.

Lysine β-hydroxybutyrylation (Kbhb) is a post-translational modification that has recently been found to regulate protein functions. However, whether and how protein Kbhb modification participates in Alzheimer's disease (AD) remains unknown. Herein, we carried out 4D label-free β-hydroxybutylation quantitative proteomics using brain samples of 8-month-old and 2-month-old APP/PS1 AD model mice and wild-type (WT) controls. We identified a series of tricarboxylic acid (TCA) cycle-associated enzymes including citrate synthase (CS) and succinate-CoA ligase subunit alpha (SUCLG1), whose Kbhb modifications were decreased in APP/PS1 mice at pathological stages. Sodium β-hydroxybutyrate (Na-β-OHB) treatment markedly increased Kbhb modifications of CS and SUCLG1 and their enzymatic activities, leading to elevated ATP production. We further found that Kbhb modifications at lysine 393 site in CS and at lysine 81 site in SUCLG1 were crucial for their enzymatic activities. Finally, we found that β-OHB levels were decreased in the brain of APP/PS1 mice at pathological stages. While ketogenic diet not only significantly increased β-OHB levels, Kbhb modifications and enzymatic activities of CS and SUCLG1, and ATP production, but also dramatically attenuated β-amyloid plaque pathologies and microgliosis in APP/PS1 mice. Together, our findings indicate the importance of protein Kbhb modification for maintaining normal TCA cycle and ATP production and provide a novel molecular mechanism underlying the beneficial effects of ketogenic diet on energy metabolism and AD intervention.

Nitrite-driven anaerobic ethane oxidation

Ethane, the second most abundant gaseous hydrocarbon in vast anoxic environments, is an overlooked greenhouse gas. Microbial anaerobic oxidation of ethane can be driven by available electron acceptors such as sulfate and nitrate. However, despite nitrite being a more thermodynamically feasible electron acceptor than sulfate or nitrate, little is known about nitrite-driven anaerobic ethane oxidation. In this study, a microbial culture capable of nitrite-driven anaerobic ethane oxidation was enriched through the long-term operation of a nitrite-and-ethane-fed bioreactor. During continuous operation, the nitrite removal rate and the theoretical ethane oxidation rate remained stable at approximately 25.0mg NO2 -N L-1d-1 and 11.48mgC2H6 L-1d-1, respectively. Batch tests demonstrated that ethane is essential for nitrite removal in this microbial culture. Metabolic function analysis revealed that a species affiliated with a novel genus within the family Rhodocyclaceae, designated as 'Candidatus Alkanivoras nitrosoreducens', may perform the nitrite-driven anaerobic ethane oxidation. In the proposed metabolic model, despite the absence of known genes for ethane conversion to ethyl-succinate and succinate-CoA ligase, 'Ca. A. nitrosoreducens' encodes a prospective fumarate addition pathway for anaerobic ethane oxidation and a complete denitrification pathway for nitrite reduction to nitrogen. These findings advance our understanding of nitrite-driven anaerobic ethane oxidation, highlighting the previously overlooked impact of anaerobic ethane oxidation in natural ecosystems.

Osmotic response in Leptospirillum ferriphilum isolated from an industrial copper bioleaching environment to sulfate

Iron and sulfur-oxidizing microorganisms play important roles in several natural and industrial processes. Leptospirillum (L.) ferriphilum, is an iron-oxidizing microorganism with a remarkable adaptability to thrive in extreme acidic environments, including heap bioleaching processes, acid mine drainage (AMD) and natural acidic water. A strain of L. ferriphilum (IESL25) was isolated from an industrial bioleaching process in northern Chile. This strain was challenged to grow at increasing concentrations of sulfate in order to assess changes in protein expression profiles, cells shape and to determine potential compatible solute molecules. The results unveiled changes in three proteins: succinyl CoA (SCoA) synthetase, isocitrate dehydrogenase (IDH) and aspartate semialdehyde dehydrogenase (ASD); which were notably overexpressed when the strain grew at elevated concentrations of sulfate. ASD plays a pivotal role in the synthesis of the compatible solute ectoine, which was identified along with hydroxyectoine by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The relationship between IDH, SCoA, and ectoine production could be due to the TCA cycle, in which both enzymes produce metabolites that can be utilized as precursors or intermediates in the biosynthesis of ectoine. In addition, distinct filamentous cellular morphology in L. ferriphilum IESL25 was observed when growing under sulfate stress conditions. This study highlights a new insight into the possible cellular responses of L. ferriphilum under the presence of high sulfate levels, commonly found in bioleaching of sulfide minerals or AMD environments.

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 40mM 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.

Identification of the succinate-CoA ligase protein gene family reveals that TaSUCL1-1 positively regulate cadmium resistance in wheat

The Succinate-CoA ligase (SUCL1) gene family is involved in energy metabolism, phytohormone signaling, and plant growth, development, and tolerance to stress. This is the first study to analyze the SUCL1 gene family in wheat (Triticum aestivum). 17 TaSUCL1 genes were identified in the complete genome sequence and classified into five subfamilies based on related genes found in three other species. The 17 TaSUCL1 genes were unevenly distributed across 11 chromosomes, and the collinearity of these genes was further investigated. Through using real-time qPCR (RT-qPCR) analysis, we identified the expression patterns of the TaSUCL1 genes under various tissues and different heavy metal stress conditions. The functions of selected TaSUCL1-1 gene were investigated by RNA interference (RNAi). This study provided a comprehensive analysis of the TaSUCL1 gene family. Within the TaSUCL1 genes, the exon-intron structure and motif composition exhibited significant similarity among members of the same evolutionary branch. Homology analysis and phylogenetic comparison of the SUCL1 genes in different plants offered valuable insights for studying the evolutionary characteristics of the SUCL1 genes. The expression levels of the TaSUCL1 genes in different tissues and under various metal stress conditions reveal its important role in plant growth and development. Gene function analysis demonstrated that TaSUCL1-1 silenced wheat plants exhibited a decrease in the total cadmium (Cd) concentrations and gene expression levels compared to the wild type (WT). Additionally, TaSUCL1-1 belonging to class c physically interacts with the β-amylase protein TaBMY1 as verified by yeast two-hybridization. This research provides a useful resource for further study of the function and molecular genetic mechanism of the SUCL1 gene family members.

SUCLG1 restricts POLRMT succinylation to enhance mitochondrial biogenesis and leukemia progression

Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.

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.

Abstract 1551: Succinyl-CoA synthetase Beta-A: A novel metastasis promoting factor in lung cancer

Abstract Changes in metabolism occur when cancer cells disseminate from primary sites and metastasize. Mitochondrial metabolism plays an essential role in various biological processes of cancer cells, but the underlying mechanism through which mitochondrial factors contribute to cancer metastasis remains largely elusive. Anoikis is a form of apoptosis induced by loss of cell adhesion, and metastatic tumor cells must be anoikis resistant to survive during circulation before forming metastatic foci in distant organs. We performed an unbiased RNAi screen using a customized mitochondrion shRNA library and identified succinyl-CoA synthetase beta-A (A-SCS, also called SUCLA2), which is known to catalyze the reversible conversion of succinyl-CoA and ADP to succinate and ATP in the TCA cycle as an essential factor that promotes the survival of disseminated cancer cells during metastasis. We demonstrate that A-SCS manages redox balance during tumor metastasis, and this metabolic contribution is independent of its role in the TCA cycle. Mechanistically, A-SCS relocates from the mitochondria to the cytosol and binds to stress granules, promoting stress granules to express the redox-scavenging enzyme catalase upon cell detachment. This mitigates oxidative stress and makes cancer cells survive through anoikis, eventually promoting metastasis. A clinical correlation study comparing primary and metastasized tumors collected from lung cancer patients demonstrated that A-SCS levels align with catalase expression as well as the metastatic progression of lung cancer. Our finding provides an academic basis for developing new treatment targets against metastasis, which strikes elevated mitochondrial metabolism during cancer cell dissemination. Citation Format: Jihoon Kang, Jung Seok Hwang, Austin C. Boese, Sydney Shuff, Jaehyun Kim, Kiyoung Eun, Sumin Kang. Succinyl-CoA synthetase Beta-A: A novel metastasis promoting factor in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1551.

Two novel SUCLA2 variants cause mitochondrial DNA depletion syndrome, type 5 in two siblings.

Mitochondrial DNA depletion syndrome (MDS), characterized by succinate-CoA ligase deficiency and loss of mitochondrial DNA (mtDNA), is caused by specific variants in nuclear genes responsible for mtDNA maintenance. SUCLA2-related mitochondrial DNA depletion syndrome, type 5 (MTDPS-5), presents as a rare, severe early progressive encephalomyopathy. This report investigates a new family exhibiting clinical manifestations of MTDPS-5 and elucidates the genetic basis of this disorder. In two affected siblings, a novel maternally inherited nonsense variant [c.1234C>T (p.Arg412*)] in the SUCLA2 gene and a unique paternally inherited indel variant (g.48569263-48571020del1758insATGA) were identified. Additionally, the siblings exhibited blood mtDNA content lower than 33% compared to age-matched controls. These findings underscore the importance of assessing SUCLA2 variants in patients with severe early progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mtDNA loss, thereby broaden the mutational spectrum of this gene.

Quantitative proteomics reveals the neurotoxicity of trimethyltin chloride on mitochondria in the hippocampus of mice

Trimethyltin chloride (TMT) is a potent neurotoxin widely used as a constituent of polyvinyl chloride plastic in the industrial and agricultural fields. However, the underlying mechanisms by which TMT leads to neurotoxicity remain elusive. In the present study, we constructed a dose and time dependent neurotoxic mouse model of TMT exposure to explore the molecular mechanisms involved in TMT-induced neurological damage. Based on this model, the cognitive ability of TMT exposed mice was assessed by the Morris water maze test and a passive avoidance task. The ultrastructure of hippocampus was analyzed by the transmission electron microscope. Subsequently, proteomics integrated with bioinformatics and experimental verification were employed to reveal potential mechanisms of TMT-induced neurotoxicity. Gene ontology (GO) and pathway enrichment analysis were done by using Metascape and GeneCards database respectively. Our results demonstrated that TMT-exposed mice exhibited cognitive disorder, and mitochondrial respiratory chain abnormality of the hippocampus. Proteomics data showed that a total of 7,303 proteins were identified in hippocampus of mice of which 224 ones displayed a 1.5-fold increase or decrease in TMT exposed mice compared with controls. Further analysis indicated that these proteins were mainly involved in tricarboxylic acid (TCA) cycle and respiratory electron transport, proteasome degradation, and multiple metabolic pathways as well as inflammatory signaling pathways. Some proteins, including succinate-CoA ligase subunit (Suclg1), NADH dehydrogenase subunit 5 (Nd5), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (Ndufa4l2) and cytochrome c oxidase assembly factor 7 (Coa7), which were closely related to mitochondrial respiratory electron transport, showed TMT dose and time dependent changes in the hippocampus of mice. Moreover, apoptotic molecules Bax and cleaved caspase-3 were up-regulated, while anti-apoptotic Bcl-2 was down-regulated compared with controls. In conclusion, our findings suggest that impairment of mitochondrial respiratory chain transport and promotion of apoptosis are the potential mechanisms of TMT induced hippocampus toxicity in mice.

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism

Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.

Mitochondrial GTP metabolism controls reproductive aging in C. elegans

Healthy mitochondria are critical for reproduction. During aging, both reproductive fitness and mitochondrial homeostasis decline. Mitochondrial metabolism and dynamics are key factors in supporting mitochondrial homeostasis. However, how they are coupled to control reproductive health remains unclear. We report that mitochondrial GTP (mtGTP) metabolism acts through mitochondrial dynamics factors to regulate reproductive aging. We discovered that germline-only inactivation of GTP- but not ATP-specific succinyl-CoA synthetase (SCS) promotes reproductive longevity in Caenorhabditis elegans. We further identified an age-associated increase in mitochondrial clustering surrounding oocyte nuclei, which is attenuated by GTP-specific SCS inactivation. Germline-only induction of mitochondrial fission factors sufficiently promotes mitochondrial dispersion and reproductive longevity. Moreover, we discovered that bacterial inputs affect mtGTP levels and dynamics factors to modulate reproductive aging. These results demonstrate the significance of mtGTP metabolism in regulating oocyte mitochondrial homeostasis and reproductive longevity and identify mitochondrial fission induction as an effective strategy to improve reproductive health.

Effects of di-(2-ethylhexyl) phthalate on growth, metabolism, and virulence of the plant pathogenic bacterium Acidovorax citrulli.

Acidovorax citrulli is a seed-borne bacterial pathogen that causes bacterial fruit blotch in cucurbits and severely affects the production of cucumbers and watermelons globally. In this study, we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on the growth, metabolism, and virulence of A. citrulli. Bacterial population was not affected by DEHP exposure; moreover, significant changes were not observed in lipid peroxidation, membrane permeability, and nucleic acid leakage. However, palmitoleic acid content was increased in the cell membrane of DEHP-exposed A. citrulli. Further, DEHP exposure increased the activity of TCA cycle-related enzymes, including α-ketoglutarate dehydrogenase and succinyl-CoA synthetase, along with increase in the content of glutamate, succinate, fumarate, and malate in TCA cycle. Additionally, total 270 genes were differentially expressed by the treatment, of which 28 genes were upregulated and 242 genes, including those related to translation, flagellum-dependent cell motility, and flagellum assembly, were downregulated. Regarding virulence traits, swimming activity was decreased in DEHP-exposed A. citrulli; however, biofilm formation was not affected in in vitro assay. Moreover, relative expression of pathogenicity genes, including hrpX and hrpG, were decreased in DEHP-exposed A. citrulli compared to that of unexposed A. citrulli. Therefore, these results suggest that DEHP accumulation in soil could potentially influence the metabolism and virulence traits of A. citrulli.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.