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Nature Chemical Biology Jan 2017Small molecules are pharmacological tools of considerable value for dissecting complex biological processes and identifying potential therapeutic interventions.... (Review)
Review
Small molecules are pharmacological tools of considerable value for dissecting complex biological processes and identifying potential therapeutic interventions. Recently, the cellular quality-control process of mitophagy has attracted considerable research interest; however, the limited availability of suitable chemical probes has restricted our understanding of the molecular mechanisms involved. Current approaches to initiate mitophagy include acute dissipation of the mitochondrial membrane potential (ΔΨ) by mitochondrial uncouplers (for example, FCCP/CCCP) and the use of antimycin A and oligomycin to impair respiration. Both approaches impair mitochondrial homeostasis and therefore limit the scope for dissection of subtle, bioenergy-related regulatory phenomena. Recently, novel mitophagy activators acting independently of the respiration collapse have been reported, offering new opportunities to understand the process and potential for therapeutic exploitation. We have summarized the current status of mitophagy modulators and analyzed the available chemical tools, commenting on their advantages, limitations and current applications.
Topics: Antimycin A; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitophagy; Molecular Structure; Oligomycins
PubMed: 28103219
DOI: 10.1038/nchembio.2287 -
Bio-protocol May 2018Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their...
Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs ( Koppenol , 2011 ; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza , 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in living cells, utilizing the Seahorse XF24 Extracellular Flux Analyzer (Figure 1). The Seahorse XF24 Extracellular Flux Analyzer continuously measures oxygen concentration and proton flux in the cell supernatant over time ( Wu , 2007 ). These measurements are converted in OCR and ECAR values and enable a direct quantification of mitochondrial respiration and glycolysis. With this protocol, we sought to assess basal mitochondrial function and mitochondrial stress of three different cancer cell lines in response to the cytotoxic test lead compound mensacarcin in order to investigate its mechanism of action. Cells were plated in XF24 cell culture plates and maintained for 24 h. Prior to analysis, the culture media was replaced with unbuffered DMEM pH 7.4 and cells were then allowed to equilibrate in a non-CO incubator immediately before metabolic flux analysis using the Seahorse XF to allow for precise measurements of Milli-pH unit changes. OCR and ECAR were measured under basal conditions and after injection of compounds through drug injection ports. With the described protocol we assess the basic energy metabolism profiles of the three cell lines as well as key parameters of mitochondrial function in response to our test compound and by sequential addition of mitochondria perturbing agents oligomycin, FCCP and rotenone/antimycin A. Figure 1.Overview of seahorse experiment.
PubMed: 34285967
DOI: 10.21769/BioProtoc.2850 -
Autophagy Nov 2021PINK1 and PRKN, which cause Parkinson disease when mutated, form a quality control mitophagy pathway that is well-characterized in cultured cells. The extent to which...
PINK1 and PRKN, which cause Parkinson disease when mutated, form a quality control mitophagy pathway that is well-characterized in cultured cells. The extent to which the PINK1-PRKN pathway contributes to mitophagy , however, is controversial. This is due in large part to conflicting results from studies using one of two mitophagy reporters: mt-Keima or mito-QC. Studies using mt-Keima have generally detected PINK1-PRKN mitophagy , whereas those using mito-QC generally have not. Here, we directly compared the performance of mito-QC and mt-Keima in cell culture and in mice subjected to a PINK1-PRKN activating stress. We found that mito-QC was less sensitive than mt-Keima for mitophagy, and that this difference was more pronounced for PINK1-PRKN mitophagy. These findings suggest that mito-QC's poor sensitivity may account for conflicting reports of PINK1-PRKN mitophagy and caution against using mito-QC as a reporter for PINK1-PRKN mitophagy. DFP: deferiprone; EE: exhaustive exercise; FBS: fetal bovine serum; OAQ: oligomycin, antimycin, and Q-VD-OPH; OMM: outer mitochondrial membrane; PBS: phosphate-buffered saline; PD: Parkinson disease; UPS: ubiquitin-proteasome system.
Topics: Animals; Autophagy; Cells, Cultured; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Fluorescent Dyes; Mice; Mice, Transgenic; Mitophagy; Protein Kinases; Ubiquitin-Protein Ligases
PubMed: 33685343
DOI: 10.1080/15548627.2021.1896924 -
Cellular and Molecular Gastroenterology... 2023Many studies have revealed crucial roles of the gut microbiota and its metabolites in liver disease progression. However, the mechanism underlying their effects on liver...
BACKGROUND & AIMS
Many studies have revealed crucial roles of the gut microbiota and its metabolites in liver disease progression. However, the mechanism underlying their effects on liver ischemia/reperfusion (I/R) injury remain largely unknown. Here, we investigate the function of gut microbiota and its metabolites in liver I/R injury.
METHODS
C57BL/6 mice was pretreated with an antibiotic cocktail. Then, we used multi-omics detection methods including 16s rRNA sequencing, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) to explore the changes of gut microbiota and metabolites in both feces and portal blood to reveal the mechanism of their protective effect in liver I/R injury.
RESULTS
We found that antibiotic pretreatment (ABX) could significantly reduce the severity of I/R-induced hepatic injury, and this effect could be transferred to germ-free mice by fecal microbiota transplantation (FMT), suggesting a protective role of the gut microbiota depletion. During I/R, the rates of serum α-ketoglutarate (αKG) production and glutamate reduction, downstream products of gut microbiota-derived glutamine, were more significant in the ABX mice. Then, we showed that αKG could promote alternative (M2) macrophage activation through oxidative phosphorylation, and oligomycin A could inhibit M2 macrophage polarization and reversed this protective effect.
CONCLUSIONS
These findings show that the gut microbiota and its metabolites play critical roles in hepatic I/R injury by modulating macrophage metabolic reprogramming. Potential therapies that target macrophage metabolism, including antibiotic therapies and novel immunometabolism modulators, can be exploited for the treatment of liver I/R injury.
Topics: Mice; Animals; Glutamine; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Chromatography, Liquid; Tandem Mass Spectrometry; Mice, Inbred C57BL; Liver; Macrophages; Reperfusion Injury; Anti-Bacterial Agents; Ischemia
PubMed: 36706918
DOI: 10.1016/j.jcmgh.2023.01.004 -
Cell Reports Jun 2019Hair plays important roles, ranging from the conservation of body heat to the preservation of psychological well-being. Hair loss or alopecia affects millions worldwide,...
Hair plays important roles, ranging from the conservation of body heat to the preservation of psychological well-being. Hair loss or alopecia affects millions worldwide, but methods that can be used to regrow hair are lacking. We report that quiescent (telogen) hair follicles can be stimulated to initiate anagen and hair growth by small molecules that activate autophagy, including the metabolites α-ketoglutarate (α-KG) and α-ketobutyrate (α-KB), and the prescription drugs rapamycin and metformin, which impinge on mTOR and AMPK signaling. Stimulation of hair growth by these agents is blocked by specific autophagy inhibitors, suggesting a mechanistic link between autophagy and hair regeneration. Consistently, increased autophagy is detected upon anagen entry during the natural hair follicle cycle, and oral α-KB prevents hair loss in aged mice. Our finding that anagen can be pharmacologically activated in telogen skin when natural anagen-inducing signal(s) are absent has implications for the treatment of hair loss patients.
Topics: AMP-Activated Protein Kinases; Aging; Allyl Compounds; Alopecia; Animals; Autophagy; Butyrates; Cell Division; Female; Hair; Hair Follicle; Ketoglutaric Acids; Male; Metformin; Mice; Mice, Inbred C57BL; Oligomycins; Quinazolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases
PubMed: 31216464
DOI: 10.1016/j.celrep.2019.05.070 -
Proceedings of the National Academy of... Jan 2021In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement...
In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including , , , , , and Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.
Topics: 3-Hydroxyacyl CoA Dehydrogenases; Activating Transcription Factor 4; Adaptor Proteins, Signal Transducing; Amino Acids; Antimycin A; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Culture Media; Gene Editing; Gene Expression Regulation; Genome, Human; Glucose; HEK293 Cells; Humans; Insulin Receptor Substrate Proteins; Mechanistic Target of Rapamycin Complex 1; Mitochondria; Neoplasm Proteins; Oligomycins; Protein Serine-Threonine Kinases; RNA, Guide, CRISPR-Cas Systems; Serine-Arginine Splicing Factors; Signal Transduction; eIF-2 Kinase
PubMed: 33483422
DOI: 10.1073/pnas.2022120118 -
ELife Sep 2022Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances...
Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating bacteria such as . By contrast, 'alternatively activated' M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here, we demonstrate that activation with IL-4 and IL-13 counterintuitively induces protective innate memory against mycobacterial challenge. In human and murine models, prior activation with IL-4/13 enhances pro-inflammatory cytokine secretion in response to a secondary stimulation with mycobacterial ligands. In our murine model, enhanced killing capacity is also demonstrated. Despite this switch in phenotype, IL-4/13 trained murine macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4/13 trained macrophages. Lastly, this work identifies that IL-10 attenuates protective IL-4/13 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.
Topics: Animals; Cytokines; Glucose; Humans; Interleukin-10; Interleukin-13; Interleukin-4; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Oligomycins; Oxidative Phosphorylation
PubMed: 36173104
DOI: 10.7554/eLife.74690 -
Redox Biology Sep 2021Mitochondrial dysfunction is a fundamental challenge in septic cardiomyopathy. Mitophagy and the mitochondrial unfolded protein response (UPR) are the predominant...
Mitochondrial dysfunction is a fundamental challenge in septic cardiomyopathy. Mitophagy and the mitochondrial unfolded protein response (UPR) are the predominant stress-responsive and protective mechanisms involved in repairing damaged mitochondria. Although mitochondrial homeostasis requires the coordinated actions of mitophagy and UPR, their molecular basis and interactive actions are poorly understood in sepsis-induced myocardial injury. Our investigations showed that lipopolysaccharide (LPS)-induced sepsis contributed to cardiac dysfunction and mitochondrial damage. Although both mitophagy and UPR were slightly activated by LPS in cardiomyocytes, their endogenous activation failed to prevent sepsis-mediated myocardial injury. However, administration of urolithin A, an inducer of mitophagy, obviously reduced sepsis-mediated cardiac depression by normalizing mitochondrial function. Interestingly, this beneficial action was undetectable in cardiomyocyte-specific FUNDC1 knockout (FUNDC1) mice. Notably, supplementation with a mitophagy inducer had no impact on UPR, whereas genetic ablation of FUNDC1 significantly upregulated the expression of genes related to UPR in LPS-treated hearts. In contrast, enhancement of endogenous UPR through oligomycin administration reduced sepsis-mediated mitochondrial injury and myocardial dysfunction; this cardioprotective effect was imperceptible in FUNDC1 mice. Lastly, once UPR was inhibited, mitophagy-mediated protection of mitochondria and cardiomyocytes was partly blunted. Taken together, it is plausible that endogenous UPR and mitophagy are slightly activated by myocardial stress and they work together to sustain mitochondrial performance and cardiac function. Endogenous UPR, a downstream signal of mitophagy, played a compensatory role in maintaining mitochondrial homeostasis in the case of mitophagy inhibition. Although UPR activation had no negative impact on mitophagy, UPR inhibition compromised the partial cardioprotective actions of mitophagy. This study shows how mitophagy modulates UPR to attenuate inflammation-related myocardial injury and suggests the potential application of mitophagy and UPR targeting in the treatment of myocardial stress.
Topics: Animals; Inflammation; Membrane Proteins; Mice; Mice, Knockout; Mitochondrial Proteins; Mitophagy; Unfolded Protein Response
PubMed: 34174558
DOI: 10.1016/j.redox.2021.102049 -
Methods in Molecular Biology (Clifton,... 2017Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The...
Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.
Topics: Adenosine Triphosphate; Animals; Antimycin A; Biological Assay; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Respiration; Cell Survival; Electron Transport Complex I; Electron Transport Complex III; Mice; Mitochondria; Muscle Fibers, Skeletal; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Primary Cell Culture; Proton Ionophores; Rotenone; Satellite Cells, Skeletal Muscle; Uncoupling Agents
PubMed: 28470519
DOI: 10.1007/978-1-4939-6960-9_7 -
Computers in Biology and Medicine Sep 2022Gliomas are malignant tumors in the central nervous system. Cuproptosis is a newly discovered cell death mechanism targeting lipoylated tricarboxylic acid cycle...
Gliomas are malignant tumors in the central nervous system. Cuproptosis is a newly discovered cell death mechanism targeting lipoylated tricarboxylic acid cycle proteins. Previous studies have found that cuproptosis participates in tumor progression, but its role in gliomas is still elusive. Here, we systematically explored the bulk-tumor and single-cell transcriptome data to reveal its role in gliomas. The cuproptosis activity score (CuAS) was constructed based on cuproptosis-related genes, and machine learning techniques validated the score stability. High CuAS gliomas were more likely to have a poor prognosis and an aggressive mesenchymal (MES) subtype. Subsequently, the SCENIC algorithm predicted 20 CuAS-related transcription factors (TFs) in gliomas. Function enrichment and microenvironment analyses found that CuAS was associated with tumor immune infiltration. Accordingly, intercellular communications between neoplasm and immunity were explored by the R package "Cellchat". Five signaling pathways and 8 ligand-receptor pairs including ICAM1, ITGAX, ITGB2, ANXA1-FRR1, and the like, were identified to suggest how cuproptosis activity connected neoplastic and immune cells. Critically, 13 potential drugs targeting high CuAs gliomas were predicted according to the CTRP and PRISM databases, including oligomycin A, dihydroartemisinin, and others. Taken together, cuproptosis is involved in glioma aggressiveness, neoplasm-immune interactions, and may be used to assist in drug selection.
Topics: Humans; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Glioma; Transcriptome; Tumor Microenvironment; Copper; Apoptosis
PubMed: 35964468
DOI: 10.1016/j.compbiomed.2022.105924