The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

Background: This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. Methods: We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. Results: We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. Conclusion: The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

Delineating the heterogeneity of senescence-induced-functional alterations in hepatocytes.

BACKGROUND AND AIM: Cellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory cytokines. This 'zombie-like' state perpetuates harmful effects on tissues and holds potential implications for liver disease progression. Remarkably, senescence exhibits heterogeneity, stemming from two crucial factors: the inducing stressor and the cell type. As such, our present study endeavors to characterize stressor-specific changes in senescence phenotype, its related molecular patterns, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs). METHODS: PMH, isolated by collagenase-perfused mouse liver (C57B6/J; 18-23 weeks), were cultured overnight in William's E-medium supplemented with 2% FBS, L-glutamine, and hepatocyte growth supplements. HepOrgs were developed by culturing cells in a 3D matrix for two weeks. The senescence was induced by DNA damage (doxorubicin, cisplatin, and etoposide), oxidative stress (H2O2, and ethanol), and telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyl transferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine and hydroxyurea). SA-ß galactosidase activity, immunofluorescence, immunoblotting, and senescence-associated secretory phenotype (SASP), and cellular bioenergetics were used to assess the senescence phenotype. RESULTS: Each senescence inducer triggers a unique combination of senescence markers in hepatocytes. All senescence inducers, except hydroxyurea and ethanol, increased SA-ß galactosidase activity, the most commonly used marker for cellular senescence. Among the SASP factors, CCL2 and IL-10 were consistently upregulated, while Plasminogen activator inhibitor-1 exhibited global downregulation across all modes of senescence. Notably, DNA damage response was activated by DNA damage inducers. Cell cycle markers were most significantly reduced by doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence shifted cellular bioenergetics capacity from glycolysis to oxidative phosphorylation. In HepOrgs exposed to senescence inducers, there was a notable increase in γH2A.X, p53, and p21 levels. Interestingly, while showing a similar trend, SASP gene expression in HepOrgs was significantly higher compared to PMH, demonstrating a several-fold increase. CONCLUSION: In our study, we demonstrated that each senescence inducer activates a unique combination of senescence markers in PMH. Doxorubicin demonstrated the highest efficacy in inducing senescence, followed by cisplatin and H2O2, with no impact on apoptosis. Each inducer prompted DNA damage response and mitochondrial dysfunction, independent of MAPK/AKT.

Mitochondrial involvement in sarcopenia.

Sarcopenia lowers the quality-of-life for millions of people across the world, as accelerated loss of skeletal muscle mass and function contributes to both age- and disease-related frailty. Physical activity remains the only proven therapy for sarcopenia to date, but alternatives are much sought after to manage this progressive muscle disorder in individuals who are unable to exercise. Mitochondria have been widely implicated in the etiology of sarcopenia and are increasingly suggested as attractive therapeutic targets to help restore the perturbed balance between protein synthesis and breakdown that underpins skeletal muscle atrophy. Reviewing current literature, we note that mitochondrial bioenergetic changes in sarcopenia are generally interpreted as intrinsic dysfunction that renders muscle cells incapable of making sufficient ATP to fuel protein synthesis. Based on the reported mitochondrial effects of therapeutic interventions, however, we argue that the observed bioenergetic changes may instead reflect an adaptation to pathologically decreased energy expenditure in sarcopenic muscle. Discrimination between these mechanistic possibilities will be crucial for improving the management of sarcopenia.

MicroRNA-29b-3p degenerates terminally differentiated dopaminergic SH-SY5Y cells by perturbation of mitochondrial functions.

Mitochondrial dysfunction is the main cause of gradual deterioration of structure and function of neuronal cells, eventually resulting in neurodegeneration. Studies have revealed a complex interrelationship between neurotoxicant exposure, mitochondrial dysfunction, and neurodegenerative diseases. Alteration in the expression of microRNAs (miRNAs) has also been linked with disruption in mitochondrial homeostasis and bioenergetics. In our recent research (Cellular and Molecular Neurobiology (2023) https://doi.org/10.1007/s10571-023-01362-4), we have identified miR-29b-3p as one of the most significantly up-regulated miRNAs in the blood of Parkinson's patients. The findings of the present study revealed that neurotoxicants of two different natures, that is, arsenic or rotenone, dramatically increased miR-29b-3p expression (18.63-fold and 12.85-fold, respectively) in differentiated dopaminergic SH-SY5Y cells. This dysregulation of miR-29b-3p intricately modulated mitochondrial morphology, induced oxidative stress, and perturbed mitochondrial membrane potential, collectively contributing to the degeneration of dopaminergic cells. Additionally, using assays for mitochondrial bioenergetics in live and differentiated SH-SY5Y cells, a reduction in oxygen consumption rate (OCR), maximal respiration, basal respiration, and non-mitochondrial respiration was observed in cells transfected with mimics of miR-29b-3p. Inhibition of miR-29b-3p by transfecting inhibitor of miR-29b-3p prior to exposure to neurotoxicants significantly restored OCR and other respiration parameters. Furthermore, we observed that induction of miR-29b-3p activates neuronal apoptosis via sirtuin-1(SIRT-1)/YinYang-1(YY-1)/peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-regulated Bcl-2 interacting protein 3-like-dependent mechanism. Collectively, our studies have shown the role of miR-29b-3p in dysregulation of mitochondrial bioenergetics during degeneration of dopaminergic neurons via regulating SIRT-1/YY-1/PGC-1α axis.

Secretory products of DPSC mitigate inflammatory effects in microglial cells by targeting MAPK pathway.

Activated microglial cells in the central nervous system (CNS) are the main contributors to neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Inhibiting their activation will help in reducing inflammation and oxidative stress during pathogenesis, potentially limiting the progression of the diseases. The immunomodulation properties of dental pulp-derived stem cells (DPSC) make it a promising therapy for neurodegenerative disorders. This study aims to determine whether secretory factors of DPSC (DPSC℗) inhibit inflammation and proliferation of microglial cells and define the molecular mechanisms. Our quantitative RT-PCR analysis showed that the DPSC℗ reduced the markers of the inflammation and induced anti-inflammatory molecules in microglial cells. DPSC ℗ reduced the intracellular and mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential in microglial cells. In addition, DPSC ℗ decreased the cellular bioenergetics parameters related to oxygen consumption rate (OCAR) and extracellular acidification rate (ECAR). We found that DPSC℗ inhibited microglial cell proliferation by activating a checkpoint molecule, Chk1 leading an arrest at the G1 phase of the cell cycle. To define the mechanism, we performed the western blot analysis and observed that the MAPK P38 pathway was inhibited by DPSC℗. Furthermore, a System biology analysis revealed that the BDNF and GDNF, secretory factors of DPSC, blocked at the phosphorylation site (Tyr 182) of the P38 molecule resulting in the inhibition of downstream signaling of inflammation. These data suggest that the DPSC℗ may be a potential therapeutic agent for neurodegenerative diseases.

The exceptional form and function of the giant bacterium Ca. Epulopiscium viviparus revolves around its sodium motive force.

Epulopiscium spp. are the largest known heterotrophic bacteria; a large cigar-shaped individual is a million times the volume of Escherichia coli. To better understand the metabolic potential and relationship of Epulopiscium sp. type B with its host Naso tonganus, we generated a high-quality draft genome from a population of cells taken from a single fish. We propose the name Candidatus Epulopiscium viviparus to describe populations of this best-characterized Epulopiscium species. Metabolic reconstruction reveals more than 5% of the genome codes for carbohydrate active enzymes, which likely degrade recalcitrant host-diet algal polysaccharides into substrates that may be fermented to acetate, the most abundant short-chain fatty acid in the intestinal tract. Moreover, transcriptome analyses and the concentration of sodium ions in the host intestinal tract suggest that the use of a sodium motive force (SMF) to drive ATP synthesis and flagellar rotation is integral to symbiont metabolism and cellular biology. In natural populations, genes encoding both F-type and V-type ATPases and SMF generation via oxaloacetate decarboxylation are among the most highly expressed, suggesting that ATPases synthesize ATP and balance ion concentrations across the cell membrane. High expression of these and other integral membrane proteins may allow for the growth of its extensive intracellular membrane system. Further, complementary metabolism between microbe and host is implied with the potential provision of nitrogen and B vitamins to reinforce this nutritional symbiosis. The few features shared by all bacterial behemoths include extreme polyploidy, polyphosphate synthesis, and thus far, they have all resisted cultivation in the lab.

Guanidinoacetic Acid Attenuates Adipogenesis through Regulation of miR-133a in Sheep.

Guanidinoacetic acid (GAA) is an amino acid derivative, previously described in the skeletal muscle of vertebrates, that serves as an important regulator of cellular bioenergetics and has been widely used as a feed additive. Nevertheless, the effect of GAA on adipose tissue growth remains unclear. Here, we hypothesized that dietary GAA negatively affected adipose tissue development in lambs. Lambs were individually fed diets with (0.09%) or without GAA for 70 d ad libitum, and the subcutaneous adipose tissues were sampled for analysis. The results showed that dietary GAA supplementation decreased the girth rib (GR) value (p < 0.01) of lamb carcasses. Both real-time PCR and Western blot analysis suggested that dietary GAA inhibited the expression of adipogenic markers, including peroxisome proliferator-activated receptor γ (PPARγ, p < 0.05), CCAAT/enhancer-binding protein α (C/EBPα, p < 0.01) and sterol-regulatory-element-binding protein 1c (SREBP1C, p < 0.01) in subcutaneous adipose tissue. In vitro, GAA inhibited sheep stromal vascular fraction (SVF) cell proliferation, which was associated with downregulation of proliferating cell nuclear antigen (PCNA, p < 0.05), cyclin-dependent kinase 4 (CDK 4, p < 0.05) and cyclin D1 (p < 0.01). GAA suppressed adipogenesis of SVF cells. Furthermore, miRNA sequencing revealed that GAA affected the miRNA expression profile, and real-time PCR analysis confirmed that miR-133a expression in both subcutaneous adipose tissue and SVF cell was downregulated by GAA. Meanwhile, miR-133a promoted adipogenic differentiation of SVF cells by targeting Sirt1. miR-133a mimics alleviated the inhibitory effect of GAA on SVF cells' adipogenic differentiation. In summary, GAA attenuated adipogenesis of sheep SVF cells, which might occur through miR-133a-modulated Sirt1 expression.

Incretin Mimetics Restore the ER-Mitochondrial Axis and Switch Cell Fate Towards Survival in LUHMES Dopaminergic-Like Neurons: Implications for Novel Therapeutic Strategies in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that afflicts more than 10 million people worldwide. Available therapeutic interventions do not stop disease progression. The etiopathogenesis of PD includes unbalanced calcium dynamics and chronic dysfunction of the axis of the endoplasmic reticulum (ER) and mitochondria that all can gradually favor protein aggregation and dopaminergic degeneration. OBJECTIVE: In Lund Human Mesencephalic (LUHMES) dopaminergic-like neurons, we tested novel incretin mimetics under conditions of persistent, calcium-dependent ER stress. METHODS: We assessed the pharmacological effects of Liraglutide-a glucagon-like peptide-1 (GLP-1) analog-and the dual incretin GLP-1/GIP agonist DA3-CH in the unfolded protein response (UPR), cell bioenergetics, mitochondrial biogenesis, macroautophagy, and intracellular signaling for cell fate in terminally differentiated LUHMES cells. Cells were co-stressed with the sarcoplasmic reticulum calcium ATPase (SERCA) inhibitor, thapsigargin. RESULTS: We report that Liraglutide and DA3-CH analogs rescue the arrested oxidative phosphorylation and glycolysis. They mitigate the suppressed mitochondrial biogenesis and hyper-polarization of the mitochondrial membrane, all to re-establish normalcy of mitochondrial function under conditions of chronic ER stress. These effects correlate with a resolution of the UPR and the deficiency of components for autophagosome formation to ultimately halt the excessive synaptic and neuronal death. Notably, the dual incretin displayed a superior anti-apoptotic effect, when compared to Liraglutide. CONCLUSIONS: The results confirm the protective effects of incretin signaling in ER and mitochondrial stress for neuronal degeneration management and further explain the incretin-derived effects observed in PD patients.

The Novel Role of Mitochondrial Citrate Synthase and Citrate in the Pathophysiology of Alzheimer's Disease.

Citrate synthase is a key mitochondrial enzyme that utilizes acetyl-CoA and oxaloacetate to form citrate in the mitochondrial membrane, which participates in energy production in the TCA cycle and linked to the electron transport chain. Citrate transports through a citrate malate pump and synthesizes acetyl-CoA and acetylcholine (ACh) in neuronal cytoplasm. In a mature brain, acetyl-CoA is mainly utilized for ACh synthesis and is responsible for memory and cognition. Studies have shown low citrate synthase in different regions of brain in Alzheimer's disease (AD) patients, which reduces mitochondrial citrate, cellular bioenergetics, neurocytoplasmic citrate, acetyl-CoA, and ACh synthesis. Reduced citrate mediated low energy favors amyloid-ß (Aß) aggregation. Citrate inhibits Aß25-35 and Aß1-40 aggregation in vitro. Hence, citrate can be a better therapeutic option for AD by improving cellular energy and ACh synthesis, and inhibiting Aß aggregation, which prevents tau hyperphosphorylation and glycogen synthase kinase-3 beta. Therefore, we need clinical studies if citrate reverses Aß deposition by balancing mitochondrial energy pathway and neurocytoplasmic ACh production. Furthermore, in AD's silent phase pathophysiology, when neuronal cells are highly active, they shift ATP utilization from oxidative phosphorylation to glycolysis and prevent excessive generation of hydrogen peroxide and reactive oxygen species (oxidative stress) as neuroprotective action, which upregulates glucose transporter-3 (GLUT3) and pyruvate dehydrogenase kinase-3 (PDK3). PDK3 inhibits pyruvate dehydrogenase, which decreases mitochondrial-acetyl-CoA, citrate, and cellular bioenergetics, and decreases neurocytoplasmic citrate, acetyl-CoA, and ACh formation, thus initiating AD pathophysiology. Therefore, GLUT3 and PDK3 can be biomarkers for silent phase of AD.

Investigating the effects of 7-ketocholesterol on retinal pigment epithelium bioenergetics.

Age-related macular degeneration (AMD) is associated with formation of drusen, clusters of lipids, and oxidized lipid products under the retinal pigment epithelium (RPE). 7-Ketocholesterol (7KC) is a form of oxidized cholesterol present in drusen and is hypothesized to play a role in AMD pathogenesis. The association of 7KC with cellular toxicity and inflammation, key elements of AMD pathology, has been demonstrated. However, the effects of 7KC on altering RPE bioenergetics, a potentially important pathologic process in AMD, are unclear. Herein, we describe the effects of non-lethal doses of 7KC on the bioenergetics and phenotype of RPE cells in culture. Metabolic analysis demonstrated a significant dose-dependent increase in total ATP production rates that was driven primarily by an increase in glycolysis. The increase in glycolysis was accompanied by an increase in glucose uptake and increased expression of hexokinase 1. Increased levels of Translocase of Outer Mitochondrial Membrane 20 and NADH:Ubiquinone Oxidoreductase Core Subunit S1, Succinate dehydrogenase, Ubiquinol-Cytochrome C Reductase Core Protein 2, Cytochrome C Oxidase II, and ATP synthase subunit beta, proteins involved in oxidative phosphorylation (OXPHOS), were also seen. However, specific electron transport chain activity remained unchanged. 7KC-treated cells also demonstrated a change in cellular morphology with decreased expression of epithelial markers. In summary, 7KC has significant effects on the bioenergetics and morphology of RPE cells reflective of findings seen in clinical AMD.

The Ethyl acetate extract from Celastrus orbiculatus suppresses non-small-cell lung cancer by activating Hippo signaling and inhibiting YAP nuclear translocation.

BACKGROUND: Celastrus orbiculatus Thunb. is a medicinal plant that has been widely used for thousands of years in China, and the ethyl acetate extract (Celastrus orbiculatus Thunb. Extract, COE) from its stem was reported to exert antitumor and anti-inflammatory effects in various preclinical studies. However, the anti-non-small-cell lung cancer activity of COE and its potential mechanism are not yet fully understood. PURPOSE: To investigate the antitumor effects of COE on non-small-cell lung cancer (NSCLC) cells and explore its molecular mechanism from the perspective of Hippo signaling, YAP nuclear translocation, and reactive oxygen species (ROS) generation. METHODS: The effects of COE on proliferation, cell cycle arrest, apoptosis, stemness, and senescence in NSCLC cell lines were determined by CCK-8, clone formation, flow cytometry, and ß-galactosidase staining assays. The effects of COE on Hippo signaling were investigated by Western blotting. The intracellular expression and distribution of YAP were analyzed by immunofluorescence assay. DCFH-DA probe combined with flow cytometry was used to detect intracellular total ROS levels in NSCLC cells after COE treatment. Xenograft tumor model was established, and the animal living image system was employed to analyze the effects of COE on the Hippo-YAP signaling in vivo. RESULT: COE significantly inhibited NSCLC activity in vitro and in vivo, mainly by proliferation inhibition, cycle arrest, apoptosis promotion, senescence promotion, and stemness downregulation. COE strongly activated Hippo signaling and inhibited YAP expression and nuclear retention. Activation of Hippo signaling induced by COE was associated with ROS-mediated phosphorylation of MOB1. CONCLUSION: This study demonstrated that COE inhibited NSCLC through activating Hippo signaling and suppressing YAP nuclear translocation, in which ROS may play a role in the phosphorylation of the MOB1 protein.

The Key Role of Mitochondrial Function in Health and Disease.

The role of mitochondrial function in health and disease has become increasingly recognized, particularly in the last two decades. Mitochondrial dysfunction as well as disruptions of cellular bioenergetics have been shown to be ubiquitous in some of the most prevalent diseases in our society, such as type 2 diabetes, cardiovascular disease, metabolic syndrome, cancer, and Alzheimer's disease. However, the etiology and pathogenesis of mitochondrial dysfunction in multiple diseases have yet to be elucidated, making it one of the most significant medical challenges in our history. However, the rapid advances in our knowledge of cellular metabolism coupled with the novel understanding at the molecular and genetic levels show tremendous promise to one day elucidate the mysteries of this ancient organelle in order to treat it therapeutically when needed. Mitochondrial DNA mutations, infections, aging, and a lack of physical activity have been identified to be major players in mitochondrial dysfunction in multiple diseases. This review examines the complexities of mitochondrial function, whose ancient incorporation into eukaryotic cells for energy purposes was key for the survival and creation of new species. Among these complexities, the tightly intertwined bioenergetics derived from the combustion of alimentary substrates and oxygen are necessary for cellular homeostasis, including the production of reactive oxygen species. This review discusses different etiological mechanisms by which mitochondria could become dysregulated, determining the fate of multiple tissues and organs and being a protagonist in the pathogenesis of many non-communicable diseases. Finally, physical activity is a canonical evolutionary characteristic of humans that remains embedded in our genes. The normalization of a lack of physical activity in our modern society has led to the perception that exercise is an "intervention". However, physical activity remains the modus vivendi engrained in our genes and being sedentary has been the real intervention and collateral effect of modern societies. It is well known that a lack of physical activity leads to mitochondrial dysfunction and, hence, it probably becomes a major etiological factor of many non-communicable diseases affecting modern societies. Since physical activity remains the only stimulus we know that can improve and maintain mitochondrial function, a significant emphasis on exercise promotion should be imperative in order to prevent multiple diseases. Finally, in populations with chronic diseases where mitochondrial dysfunction is involved, an individualized exercise prescription should be crucial for the "metabolic rehabilitation" of many patients. From lessons learned from elite athletes (the perfect human machines), it is possible to translate and apply multiple concepts to the betterment of populations with chronic diseases.

Crosstalk Between miRNA and Protein Expression Profiles in Nitrate-Exposed Brain Cells.

Growing evidence reported a strong association between the nitrate ingestion and adverse health consequences in humans, including its detrimental impact on the developing brain. The present study identified miRNAs and proteins in SH-SY5Y human neuroblastoma cells and HMC3 human microglial cells using high-throughput techniques in response to nitrate level most prevalent in the environment (India) as X dose and an exceptionally high nitrate level as 5X dose that can be reached in the near future. Cells were exposed to mixtures of nitrates for 72 h at doses of X and 5X, 320 mg/L and 1600 mg/L, respectively. OpenArray and LCMS analysis revealed maximum deregulation in miRNAs and proteins in cells exposed to 5X dose. Top deregulated miRNAs include miR-34b, miR-34c, miR-155, miR-143, and miR-145. The proteomic profiles of both cell types include proteins that are potential targets of deregulated miRNAs. These miRNAs and their targeted proteins involve in multiple functions, including metabolic processes, mitochondrial functions, autophagy, necroptosis, apoptosis, neuronal disorders, brain development, and homeostasis. Furthermore, measuring mitochondrial bioenergetics in cells exposed to nitrate revealed that a 5X dose causes a significant reduction in oxygen consumption rate (OCR) and other bioenergetic parameters in both cell types. In summary, our studies have demonstrated that a 5X dose of nitrate significantly alters cellular physiology and functions by deregulating several miRNAs and proteins. However, X dose of nitrate has not caused any adverse effects on any cell type.

The Human Mercaptopyruvate Sulfurtransferase TUM1 Is Involved in Moco Biosynthesis, Cytosolic tRNA Thiolation and Cellular Bioenergetics in Human Embryonic Kidney Cells.

Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics.

NADPH oxidase 4 is dispensable for skin myofibroblast differentiation and wound healing.

Differentiation of fibroblasts to myofibroblasts is governed by the transforming growth factor beta (TGF-ß) through a mechanism involving redox signaling and generation of reactive oxygen species (ROS). Myofibroblasts synthesize proteins of the extracellular matrix (ECM) and display a contractile phenotype. Myofibroblasts are predominant contributors of wound healing and several pathological states, including fibrotic diseases and cancer. Inhibition of the ROS-generating enzyme NADPH oxidase 4 (NOX4) has been proposed to mitigate fibroblast to myofibroblast differentiation and to offer a therapeutic option for the treatment of fibrotic diseases. In this study, we addressed the role of NOX4 in physiological wound healing and in TGF-ß-induced myofibroblast differentiation. We explored the phenotypic changes induced by TGF-ß in primary skin fibroblasts isolated from Nox4-deficient mice by immunofluorescence, Western blotting and RNA sequencing. Mice deficient for Cyba, the gene coding for p22phox, a key subunit of NOX4 were used for confirmatory experiments as well as human primary skin fibroblasts. In vivo, the wound healing was similar in wild-type and Nox4-deficient mice. In vitro, despite a strong upregulation following TGF-ß treatment, Nox4 did not influence skin myofibroblast differentiation although a putative NOX4 inhibitor GKT137831 and a flavoprotein inhibitor diphenylene iodonium mitigated this mechanism. Transcriptomic analysis revealed upregulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3 in Nox4-deficient fibroblasts, which had however no impact on fibroblast bioenergetics. Altogether, we provide extensive evidence that NOX4 is dispensable for wound healing and skin fibroblast to myofibroblast differentiation, and suggest that another H2O2-generating flavoprotein drives this mechanism.

Plasticity of cancer invasion and energy metabolism.

Energy deprivation is a frequent adverse event in tumors that is caused by mutations, malperfusion, hypoxia, and nutrition deficit. The resulting bioenergetic stress leads to signaling and metabolic adaptation responses in tumor cells, secures survival, and adjusts migration activity. The kinetic responses of cancer cells to energy deficit were recently identified, including a switch of invasive cancer cells to energy-conservative amoeboid migration and an enhanced capability for distant metastasis. We review the energy programs employed by different cancer invasion modes including collective, mesenchymal, and amoeboid migration, as well as their interconversion in response to energy deprivation, and we discuss the consequences for metastatic escape. Understanding the energy requirements of amoeboid and other dissemination strategies offers rationales for improving therapeutic targeting of metastatic cancer progression.

Secretome of Dental Pulp-Derived Stem Cells Reduces Inflammation and Proliferation of Glioblastoma Cells by Deactivating Mapk-Akt Pathway.

Background: Dental pulp-derived stem cells (DPSC) is a promising therapy as they modulate the immune response, so we evaluated the inhibitory effect of DPSC secretome (DPSC℗) on the proliferation and inflammation in human glioblastoma (GBM) cells (U-87 MG) and elucidated the concomitant mechanisms involved. Methods: The U87-MG cells were cultured with DPSC℗ for 24 h and assessed the expression of inflammatory molecules using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), generation of reactive oxygen species (ROS), and mitochondrial functionality using a seahorse flux analyzer. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay and cell cycle analysis were performed to evaluate the proliferation and cell cycle. Finally, the protein levels were determined by western blot. Results: DPSC℗ reduced the inflammation and proliferation of U-87 MG cells by down-regulating the pro-inflammatory markers and up-regulating anti-inflammatory markers expressions through ROS-mediated signaling. Moreover, DPSC℗ significantly reduced the mitochondrial membrane potential (MMP) in the cells. The cellular bioenergetics revealed that all the parameters of oxygen consumption rate (OCAR) and the extracellular acidification rate (ECAR) were significantly decreased in the GBM cells after the addition of DPSC℗. Additionally, DPSC℗ decreased the GBM cell proliferation by arresting the cell cycle at the G1 phase through activation (phosphorylation) of checkpoint molecule CHK1. Furthermore, mechanistically, we found that the DPSC℗ impedes the phosphorylation of the mitogen-activated protein kinases (P38 MAPK) and protein kinase B (AKT) pathway. Conclusion: Our findings lend the first evidence of the inhibitory effects of DPSC℗ on proliferation and inflammation in GBM cells by altering the P38 MAPK-AKT pathway.

Intracellular Injection of Brain Extracts from Alzheimer's Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics.

Strong evidence indicates that amyloid beta (Aß) inflicts its toxicity in Alzheimer's disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aß42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aßs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aß oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extract's ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aß oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.

Treatment of VLCAD-Deficient Patient Fibroblasts with Peroxisome Proliferator-Activated Receptor δ Agonist Improves Cellular Bioenergetics.

Background: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disease that prevents the body from utilizing long-chain fatty acids for energy, most needed during stress and fasting. Symptoms can appear from infancy through childhood and adolescence or early adulthood, and include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. REN001 is a peroxisome-proliferator-activated receptor delta (PPARδ) agonist that modulates the expression of the genes coding for fatty acid ß-oxidation enzymes and proteins involved in oxidative phosphorylation. Here, we assessed the effect of REN001 on VLCAD-deficient patient fibroblasts. Methods: VLCAD-deficient patient and control fibroblasts were treated with REN001. Cells were harvested for gene expression analysis, protein content, VLCAD enzyme activity, cellular bioenergetics, and ATP production. Results: VLCAD-deficient cell lines responded differently to REN001 based on genotype. All cells had statistically significant increases in ACADVL gene expression. Small increases in VLCAD protein and enzyme activity were observed and were cell-line- and dose-dependent. Even with these small increases, cellular bioenergetics improved in all cell lines in the presence of REN001, as demonstrated by the oxygen consumption rate and ATP production. VLCAD-deficient cell lines containing missense mutations responded better to REN001 treatment than one containing a duplication mutation in ACADVL. Discussion: Treating VLCAD-deficient fibroblasts with the REN001 PPARδ agonist results in an increase in VLCAD protein and enzyme activity, and a decrease in cellular stress. These results establish REN001 as a potential therapy for VLCADD as enhanced expression may provide a therapeutic increase in total VLCAD activity, but suggest the need for mutation-specific treatment augmented by other treatment measures.