Respiratory Enzymes Research Articles

From respiratory supercomplexes to megacomplexes

Cellular energy is produced in mitochondria by respiratory enzymes in a process called oxidative phosphorylation. Respiratory enzymes are large proteins embedded in the inner mitochondrial membrane which can occur either as individual enzymes or organize into larger assemblies called super- and megacomplexes. Originally, supercomplex organization was not generally accepted, but nowadays, their existence is clear. However, the functional aspects are still in the dark. In this overview, we will briefly touch upon the history of supercomplex research, the variation of the supercomplex compositions and shed light on the challenges of studying their functional aspects.

Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease.

Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024;96:1209-1224.

Intermittent episodes of acute severe encephalomyopathy and early death in two siblings caused by biallelic likely pathogenic variants in FASTKD2: Expanding phenotype and literature review.

Combined oxidative phosphorylation (OXPHOS) deficiency 44 (COXPD44; MIM# 618855) is caused by biallelic pathogenic variants in FAS-activated serine-threonine kinase domain 2 (FASTKD2) (MIM# 612322). COXPD44 is characterized by variable clinical features-developmental delay, chronic epileptic encephalopathy, seizure disorder/status epilepticus and cerebellar ataxia. We ascertained one sibwith episodic acute encephalomyopathy triggered by acute gastroenteritis and associated with haematological abnormalities, rhabdomyolysis leading to acute kidney injury, hypotensive shock leading to early death and a similarly affected sib with early death. Both siblings were normal neurologically in between the acute episodes. Whole exome sequencing (WES) was performed in the elder sibling. Mitochondrial respiratory chain enzyme activity assaywas performed in fibroblast cells and muscle tissue of the elder sibling. Also, Adenosine triphosphate (ATP) determination assay was done in fibroblast cells of the elder sibling. WES revealed compound heterozygous missense likely pathogenic variants in FASTKD2. Mitochondrial respiratory chain enzyme activity in muscle tissue showed reduced complex IV activity and ATP determination assay showed a reduction of ATP in skin fibroblasts. Herein, we report two siblings with novel clinical phenotype associated with COXPD44. Our report further validates the biallelic variants in FASTKD2 associated with the variable phenotypes and mitochondrial OXPHOS defect.

Preparation and evaluation of an active modified oxygen/ carbon dioxide preservative for packaged fresh-cut durian

In this study, a series of chitosan/copper nanocomposites (CS/Cu-NCs) served as catalyzers for VC to remove O2 in food packages were successfully synthesized, characterized, and further utilized to prepare an active modified O2/CO2 preservative. Transmission electron microscope, ultraviolet-visible, and infrared spectra confirmed CS/Cu-NCs #1–3 were successfully synthesized. Three components of the preservative were optimized, and the best formulation was: CS/Cu-NCs #2: VC: NaHCO3=0.5:1.5:0.15. It maintained relatively stable O2/CO2 in the package at 10℃. Fresh-cut durian treated with 0 g (CK), 1.15 g (T1), 2.3 g (T2), and 3.45 g (T3) preservatives were stored at 10℃. Results showed that T1 group exhibited significantly (P<0.05) higher firmness and VC content compared with CK at 12 d, indicating the best qualities. Key respiratory enzyme analyses verified that decreasing SDH, increasing G-6-PDH and 6-PGDH contributed mostly to the superior qualities of T1 group. This study could provide a prospective solution for packaged fresh-cut durian.

Effect of alternative oxidase (AOX) expression on mouse cerebral mitochondria bioenergetics

Alternative oxidase (AOX) is an enzyme that transfers electrons from reduced quinone directly to oxygen without proton translocation. When AOX from Ciona intestinalis is xenotopically expressed in mice, it can substitute the combined electron-transferring activity of mitochondrial complexes III/IV. Here, we used brain mitochondria from AOX-expressing mice with such a chimeric respiratory chain to study respiratory control bioenergetic mechanisms.AOX expression did not compromise the function of the mammalian respiratory chain at physiological conditions, however the complex IV inhibitor cyanide only partially blocked respiration by AOX-containing mitochondria. The relative fraction of cyanide-insensitive respiration increased at lower temperatures, indicative of a temperature-controlled attenuation of mammalian respiratory enzyme activity.As AOX does not translocate protons, the mitochondrial transmembrane potential in AOX-containing mitochondria was more sensitive to cyanide during succinate oxidation than during malate/pyruvate-supported respiration. High concentrations of cyanide fully collapsed membrane potential during oxidation of either succinate or glycerol 3-phosphate, but not during malate/pyruvate-supported respiration. This confirms AOX's electroneutral redox activity and indicates differences in the proton-translocating capacity of dehydrogenases upstream of the ubiquinone pool. Our respiration data refutes previous proposals for quinone partitioning within the supercomplexes of the respiratory chain, instead supporting the concept of a single homogeneous, freely diffusing quinone pool.Respiration with either succinate or glycerol 3-phosphate promotes reverse electron transfer (RET) towards complex I. AOX expression significantly decreased RET-induced ROS generation, with the effect more pronounced at low temperatures. Inhibitor-sensitivity analysis showed that the AOX-induced decrease in H2O2 release is due to the lower contribution of complex I to net ROS production during RET.Overall, our findings provide new insights into the role of temperature as a mechanism to control respiration and highlight the utility of AOX as a genetic tool to characterize both the distinct pathways of oxygen reduction and the role of redox control in RET.

Unveiling the effects of ZnO nanoparticle incorporated chitosan coating on postharvest quality of eggplant (Solanum melongena L.)

Due to rapid moisture loss, eggplant (Solanum melongena L.), a significant economic product, has a short postharvest shelf life. This study examined at 4 °C for 20 days at the effects of chitosan (CH) 1% and CH with micro ZnO (0.25 g/L) on eggplant quality. After 0, 5, 10, 15, and 20 days, coating effects on parameters (weight loss, pH, respiration rate, total acidity, soluble solids, firmness, total anthocyanin, antioxidant, phenolic, antioxidant enzymes superoxide dismutase, peroxidase and malondialdehyde activity), as well as microbiological count (total bacteria and yeast), were examined. When compared to the control, the coatings significantly affected the quality parameters, eggplant fruit coatings with CH (1 %) + nano ZnO (0.25 g/L) had the significantly least respiratory rate, weight loss, firmness, TAC, TPC, TAA and enzyme activity (P < 0.05). The microbial load during fruit preservation was suppressed by nano ZnO treatments (total bacterial and fungal count in control and eggplant treated with nano ZnO were 4.46, 4.33 and 3.96, 3.17 log CFU g−1 respectively). Treatments had little effect on fruit's flavour or aroma, but ZnO-treated fruit had improved texture, colour, and acceptance overall compared to control fruit. Finally, nano ZnO may offer a chance to preserve quality and limit deterioration during extended storage.

Facilitating Intracellular Electron Bifurcation by Mediating Flavin-Based Extracellular and Transmembrane Electron Transfer: A Novel Role of Pyrogenic Carbon in Dark Fermentation for Hydrogen Production.

Pyrogenic carbon is considered an enhancer to H2-yielding dark fermentation (DF), but little is known about how it regulates extracellular electron transfer (EET) and influences transmembrane respiratory chains and intracellular metabolisms. This study addressed these knowledge gaps and demonstrated that wood waste pyrogenic carbon (biochar) could significantly improve the DF performance; e.g., addition of pyrogenic carbon produced by pyrolysis at 800 °C (PC800) increased H2 yield by 369.7%. Biochemical quantification, electrochemical analysis, and electron respiratory chain inhibition tests revealed that PC800 promoted the extracellular flavin-based electron transfer process and further activated the acceleration of the transmembrane electron transfer. Comparative metagenome/metatranscriptome analyses indicated that the flavin-containing Rnf complex was the potential transmembrane respiratory enzyme associated with PC800-mediated EET. Based on NADH/NAD+ circulation, the promoted Rnf complex could stimulate the functions of the electron bifurcating Etf/Bcd complex and startup of glycolysis. The promoted Etf/Bcd could further contribute to balance the NADH/NAD+ level for glycolytic reactions and meanwhile provide reduced ferredoxin for group A1 [FeFe]-hydrogenases. This proton-energy-linked mechanism could achieve coupling production of ATP and H2. This study verified the important roles of pyrogenic carbon in mediating EET and transmembrane/intracellular pathways and revealed the crucial roles of electron bifurcation in DF for hydrogen production.

Decomposition of Hydrogen Peroxide in Presence of DimethylglyoximatoNickel Complexes as Catalysts: Catalase-Like Activity

The development in coordination chemistry in recent years raise hopes that synthetically produced metal complexes could mimic many biochemical systems widely found in nature. There is a certain analogy between nature and organometallic systems. A large number of biological metal complexes are known, including oxygen carriers like hemoglobin in the blood, which contains a ferrous ion; respiratory enzymes; those involved in protein hydrolysis; and vitamin B12, which is only active in the presence of cobalt in the trivalent state. The Nickel (III), in addition to Fe-S clusters, was an essential component in hydrogenases. Since then, nickel (III) complexes have been used as models for studying the catalytic function of certain enzymes (hydrogenases). In this context, a study on the catalytic ability of dimethylglyoximato-nickel complexes as peroxiredoxases in the dismutation or oxidation of hydrogen peroxide was conducted. The results were discussed, commented upon, and a reaction mechanism was proposed. The results seem encouraging, regarding the effect of the complexation on catalase- like activity.

Repeated marine heatwaves aggravate the adverse effects of nano-TiO2 on physiological metabolism of the thick-shelled mussel Mytilus coruscus

Global climate change is a major trigger of unexpected temperature fluctuations. The impacts of marine heatwaves (MHWs) and nano-titanium dioxide (nano-TiO2) on marine organisms have been extensively investigated. However, the potential mechanisms underlying their interactive effects on physiological processes and metabolism remain poorly understood, especially regarding periodic MHWs in real-world conditions. In this study, the effects of nano-TiO2 (at concentrations of 0, 25, and 250 μg/L) and periodic MHWs on the condition index (CI) and underlying metabolic mechanisms were investigated in mussels (Mytilus coruscus). The results showed that mussels try to upregulate their respiration rate (RR) to enhance aerobic metabolism (indicated by elevated succinate dehydrogenase) under short-term nano-TiO2 exposure. However, even at ambient concentration (25 μg/L), prolonged nano-TiO2 exposure inhibited ingestion ability (decreased clearance rate) and glycolysis (inhibited pyruvate kinase, hexokinase, and phosphofructokinase activities), which led to an insufficient energy supply (decreased triglyceride, albumin, and ATP contents). Repeated thermal scenarios caused more severe physiological damage, demonstrating that mussels are fragile to periodic MHWs. MHWs decreased the zeta potential of the nano-TiO2 particles but increased the hydrodynamic diameter. Additionally, exposure to nano-TiO2 and periodic MHWs further affected aerobic respiration (inhibited lactate dehydrogenase and succinate dehydrogenase activities), metabolism (decreased RR, activities of respiratory metabolism-related enzymes, and expressions of PEPCK, PPARγ, and ACO), and overall health condition (decreased ATP and CI). These findings indicate that the combined stress of these two stressors exerts more detrimental impact on the physiological performance and energy metabolism of mussels, and periodic MHWs exacerbate the toxicological effects of ambient concentration nano-TiO2. Given the potential worsening of nanoparticle pollution and the increase in extreme heat events in the future, the well-being of mussels in the marine environment may face further threats.

Influence of Water and Fertilizer Reduction on Respiratory Metabolism in Sugar Beet Taproot (Beta vulgaris L.).

Inner Mongolia, a major region in China for growing sugar beet, faces challenges caused by unscientific water and fertilizer management. This mismanagement restricts the improvement of sugar beet yield and quality and exacerbates water waste and environmental pollution. This study aims to evaluate the effects of reduced water and fertilizer on the growth and physiological metabolism of sugar beet taproot. Field experiments were conducted in Ulanqab, Inner Mongolia, in 2022 and 2023, using a split-plot design with three levels each of fertilization and irrigation. The study analyzed the effects of reduced water and fertilizer treatments on fresh taproot weight, respiration rate, energy metabolism, respiratory enzyme activity, and gene expression in sugar beet taproot. It was found that a 10% reduction in fertilizer significantly increased the beet taproot fresh weight. Further research revealed that during the rapid leaf growth phase and the taproot and sugar growth period, a 10% reduction in fertilizer upregulated HK and IDH gene expression and downregulated G6PDH gene expression in the beet taproot. This increased HK and IDH activities, decreased G6PDH activity, enhanced the activity of the EMP-TCA pathway, and inhibited the PPP. Taproot weight was positively correlated with the respiration rate, ATP content, EC, and ATPase, HK, and IDH activities, thereby increasing the taproot growth rate and taproot fresh weight, with an average increase of 4.0% over two years. These findings introduce a novel method for optimizing fertilizer use, particularly beneficial in water-scarce regions. Implementing this strategy could help farmers in western Inner Mongolia and similar areas improve crop yield and sustainability. This study offers new insights into resource-efficient agricultural practices, highlighting the importance of customized fertilization strategies tailored to local environmental conditions.

Impaired energy metabolism and altered brain histoarchitecture characterized by inhibition of glycolysis and mitochondrial electron transport-linked enzymes in rats exposed to diisononyl phthalate

The brain is an energy demanding organ, constituting about 20 % of the body's resting metabolic rate. An efficient energy metabolism is critical to neuronal functions. Glucose serves as the primary essential energy source for the adult brain and plays a critical role in supporting neural growth and development. Endocrine disrupting chemicals (EDCs) such as phthalates has been shown to have a negative impact on neurological functions. The impact of diisononyl phthalate (DiNP) on neural energy transduction using cellular energy metabolizing enzymes as indicators was examined. Over the course of 14 days, eighteen (18) albino rats divided into three groups (1,2 and 3) of six albino rats were given Tween-80/saline, 20 and 200 mg/kg body weight respectively. In the brain, we assessed histological changes as well as activities of selected enzymes of energy metabolism such as the glycolytic pathway, citric acid cycle and mitochondrial electron transport-linked complexes. Activities of the glycolytic and TCA cycle enzymes assayed were significantly decreased except citrate synthase activity with no statistically significant change following the administration of DiNP. Also, respiratory chain complexes (Complex I-IV) activities were significantly reduced when compared to control. DiNP exposure altered the histological integrity of various brain sections. These include degenerated Purkinje neurons, distortion of the granular layer and Purkinje cell layer. Data from this study indicated impaired brain energy metabolism via down-regulation of enzymes of cellular respiration of the glycolytic and oxidative phosphorylation pathways and altered brain histoarchitecture orchestrated by DiNP exposure.

Proteomic changes orchestrate metabolic acclimation of a unicellular diazotrophic cyanobacterium during light-dark cycle and nitrogen fixation states.

Cyanobacteria have developed an impressive array of proteins and pathways, each tailored for specific metabolic attributes, to execute photosynthesis and biological nitrogen (N2)-fixation. An understanding of these biologically incompatible processes provides important insights into how they can be optimized for renewable energy. To expand upon our current knowledge, we performed label-free quantitative proteomic analysis of the unicellular diazotrophic cyanobacterium Crocosphaera subtropica ATCC 51142 grown with and without nitrate under 12-hour light-dark cycles. Results showed significant shift in metabolic activities including photosynthesis, respiration, biological nitrogen fixation (BNF), and proteostasis to different growth conditions. We identified 14 nitrogenase enzymes which were among the most highly expressed proteins in the dark under nitrogen-fixing conditions, emphasizing their importance in BNF. Nitrogenase enzymes were not expressed under non nitrogen fixing conditions, suggesting a regulatory mechanism based on nitrogen availability. The synthesis of key respiratory enzymes and uptake hydrogenase (HupSL) synchronized with the synthesis of nitrogenase indicating a coordinated regulation of processes involved in energy production and BNF. Data suggests alternative pathways that cells utilize, such as oxidative pentose phosphate (OPP) and 2-oxoglutarate (2-OG) pathways, to produce ATP and support bioenergetic BNF. Data also indicates the important role of uptake hydrogenase for the removal of O2 to support BNF. Overall, this study expands upon our knowledge regarding molecular responses of Crocosphaera 51142 to nitrogen and light-dark phases, shedding light on potential applications and optimization for renewable energy.

CORRECTION OF HEALTH DEVIATIONS CAUSED BY UNFAVORABLE ENVIRONMENTAL CONDITIONS USING NON-PHARMACOLOGICAL MEANS

Lack of atmospheric oxygen (exogenous hypoxia) in residents of high mountain areas leads to functional disturbances of blood circulation, blood composition, activity of tissue respiration enzymes and resulting other biochemical abnormalities, causing a number of diseases. And the open mines operating in these regions create an unfavorable ecological environment, contributing to the deepening of functional disorders of the body among the population. Adolescents in this region are also at risk for the development of pathological processes. These risks can be reduced, and diseases can be prevented using non-drug means, using herbs with antihypoxic effects and special breathing exercises.

Antibacterial mechanism of CO2 combined with low temperature against Shewanella putrefaciens by biochemical and metabolomics analysis

To further reveal the inhibition mechanism of carbon dioxide (CO2) on Shewanella putrefaciens (S. putrefaciens), influence on metabolic function was studied by biochemical and metabolomics analysis. Accordingly, reduction of intracellular pH (pHi), depolarization of cell membrane and accumulation of reactive oxygen species (ROS) indicated that CO2 changed the membrane permeability of S. putrefaciens. Besides, adenosine triphosphate (ATP), ATPase, nicotinamide adenine dinucleotide (NAD+/NADH) and ratios of NADH/NAD+ were detected, indicating a role of CO2 in repressing respiratory pathway and electron transport. According to metabolomics results, CO2 induced differential expressions of metabolites, disordered respiratory chain and weakened energy metabolism of S. putrefaciens. Inhibition of respiratory rate-limiting enzymes also revealed that electron transfer of respiratory chain was blocked, cell respiration was weakened, and thus energy supply was insufficient under CO2 stress. These results revealed that CO2 caused disruption of metabolic function, which might be the main cause of growth inhibition for S. putrefaciens.

The two alternative NADH:quinone oxidoreductases from Staphylococcus aureus: two players with different molecular and cellular roles.

Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently. The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow S. aureus to live and survive. Despite its prominence as a pathogen, the energy metabolism of S. aureus remains underexplored, with its respiratory enzymes often escaping thorough investigation. S. aureus bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of S. aureus to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.

Alterations in coenzyme Q10 status in a cybrid line harboring the 3243A>G mutation of mitochondrial DNA is associated with abnormal mitochondrial bioenergetics and dysregulated mitochondrial biogenesis

Mitochondrial DNA (mtDNA) mutations, including the m.3243A>G mutation that causes mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with secondary coenzyme Q10 (CoQ10) deficiency. We previously demonstrated that PPARGC1A knockdown repressed the expression of PDSS2 and several COQ genes. In the present study, we compared the mitochondrial function, CoQ10 status, and levels of PDSS and COQ proteins and genes between mutant cybrids harboring the m.3243A>G mutation and wild-type cybrids. Decreased mitochondrial energy production, defective respiratory function, and reduced CoQ10 levels were observed in the mutant cybrids. The ubiquinol-10:ubiquinone-10 ratio was lower in the mutant cybrids, indicating blockage of the electron transfer upstream of CoQ, as evident from the reduced ratio upon rotenone treatment and increased ratio upon antimycin A treatment in 143B cells. The mutant cybrids exhibited downregulation of PDSS2 and several COQ genes and upregulation of COQ8A. In these cybrids, the levels of PDSS2, COQ3-a isoform, COQ4, and COQ9 were reduced, whereas those of COQ3-b and COQ8A were elevated. The mutant cybrids had repressed PPARGC1A expression, elevated ATP5A levels, and reduced levels of mtDNA-encoded proteins, nuclear DNA-encoded subunits of respiratory enzyme complexes, MNRR1, cytochrome c, and DHODH, but no change in TFAM, TOM20, and VDAC1 levels. Alterations in the CoQ10 level in MELAS may be associated with mitochondrial energy deficiency and abnormal gene regulation. The finding of a reduction in the ubiquinol-10:ubiquinone-10 ratio in the MELAS mutant cybrids differs from our previous discovery that cybrids harboring the m.8344A>G mutation exhibit a high ubiquinol-10:ubiquinone-10 ratio.

The monotopic quinone reductases from Staphylococcus aureus

Staphylococcus aureus, a Gram-positive bacterium, is an opportunistic pathogen and one of the most frequent causes for community acquired and nosocomial infections that has become a major public health threat due to the increased incidence of its drug resistance. Although being a prominent pathogen, its energetic metabolism is still underexplored, and its respiratory enzymes have been escaping attention. S. aureus can adapt to different environmental conditions by performing both aerobic and anaerobic respirations, which is particularly important as it frequently colonizes niches with different oxygen concentrations. This adaptability is derived from the composition of its respiratory chain, specifically from the presence of terminal electron acceptor reductases. The plasticity of S. aureus energy metabolism is enlarged by the ten quinone reductases encoded in its genome, eight of them being monotopic proteins. The role of these proteins is critical as they connect the different catabolic pathways to the respiratory chain. In this work, we identify, describe, and revise the monotopic quinone reductases present in S. aureus, providing an integrated view of its respiratory chain.

Effect of hydroxy-α-sanshool on lipid metabolism in liver and hepatocytes based on AMPK signaling pathway

BackgroundWith the increasing awareness of the safety of traditional Chinese medicine and food, as well as in-depth studies on the pharmacological activity and toxicity of Zanthoxylum armatum DC. (ZADC), it has been found that ZADC is hepatotoxic. However, the toxic substance basis and mechanism of action have not been fully elucidated. Hydroxy-α-sanshool (HAS) belongs to an amide compound in the fruits of ZADC, which may be hepatotoxic. However, the specific effects of HAS, including liver toxicity, are unclear. PurposeThe objectives of this research was to determine how HAS affects hepatic lipid metabolism, identify the mechanism underlying the accumulation of liver lipids by HAS, and offer assurances on the safe administration of HAS. MethodsAn in vivo experiment was performed by gavaging C57 BL/6 J mice with various dosages of HAS (5, 10, and 20 mg/kg). Biochemical indexes were measured, and histological analysis was performed to evaluate HAS hepatotoxicity. Hepatic lipid levels were determined using lipid indices and oil red O (ORO) staining. Intracellular lipid content were determined by biochemical analyses and ORO staining after treating HepG2 cells with different concentrations of HAS in vitro. Mitochondrial membrane potential, respiratory chain complex enzymes, and ATP levels were assessed by fluorescence labeling of mitochondria. The levels of proteins involved in lipogenesis and catabolism were determined using Western blotting. ResultsMice in the HAS group had elevated alanine and aspartate aminotransferase blood levels as well as increased liver index compared with the controls. The pathological findings showed hepatocellular necrosis. Serum and liver levels of triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels were increased, whereas high-density lipoprotein cholesterol levels decreased. The ORO staining findings demonstrated elevated liver lipid levels. In vitro experiments demonstrated a notable elevation in triglyceride and total cholesterol levels in the HAS group. ATP, respiratory chain complex enzyme gene expression, mitochondrial membrane potential, and mitochondrial number were reduced in the HAS group. The levels of lipid synthesis-associated proteins (ACC, FASN, and SREBP-1c) were increased, and lipid catabolism-associated protein levels (PPARα and CPT1) and the p-AMPK/AMPK ratio were decreased in vivo and in vitro. ConclusionHAS has hepatotoxic effects, which can induce fatty acid synthesis and mitochondrial function damage by inhibiting the AMPK signaling pathway, resulting in aberrant lipid increases.

Identification of proteins involved in intracellular ubiquinone trafficking in Saccharomyces cerevisiae using artificial ubiquinone probe

Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ6, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ6-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the machinery involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been demonstrated. To gain insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ6-deficient S. cerevisiae (Δcoq2) on a non-fermentable carbon source, indicating that this UQ was taken up and delivered to mitochondria, and served as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). The physiological relevance of UQ binding to these proteins is discussed.

ETMR-20. CARDIOLIPIN ACYL CHAIN MODULATION AS A NOVEL MITOCHONDRIAL-FOCUSED THERAPEUTIC TARGET IN ETMR

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive CNS neoplasm that occurs almost exclusively in infants and is associated with an extremely poor prognosis. Dysregulated mitochondrial bioenergetics and dynamics have been associated with the initiation and progression of diverse cancers and studies have linked metabolic rewiring to chemotherapy resistance. Cardiolipins are mitochondrial-specific lipids that reside in the mitochondrial membrane and their fatty acid composition has been extensively shown to regulate mitochondrial function and dynamics. Despite the known functional significance of cardiolipin structure, their role in mitochondrial regulation of brain tumors remains ill-defined. Using mass spectrometry imaging, we identified a shift to shorter acyl chain cardiolipins within the rapidly proliferating embryonal tumor cells in patient samples and patient-derived cell lines grown as 3D tumorspheres. Western blot analysis of the enzymes involved in cardiolipin synthesis and remodeling identified a significant increase in the expression of the cardiolipin remodeling enzyme, LCLAT1/ALCAT1. Orthogonal imaging techniques including immunohistochemistry, transmission electron microscopy and super-resolution microscopy correlated shorter acyl chain remodeling of cardiolipin with fragmented mitochondria (increased fission) and aberrant cristae structure. Further studies identified increased expression of the fission protein Drp1, decreased expression of respiratory chain enzymes and altered respiration in the embryonal tumor cells. Ongoing studies will utilize multiple methods to modulate cardiolipin acyl chain structure and use MS and MSI to correlate cardiolipin fatty acid structure to mitochondrial function and growth inhibition in the embryonal tumor cells.