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Autophagy Mar 2020Mitophagy, which is a conserved cellular process for selectively removing damaged or unwanted mitochondria, is critical for mitochondrial quality control and the...
Mitophagy, which is a conserved cellular process for selectively removing damaged or unwanted mitochondria, is critical for mitochondrial quality control and the maintenance of normal cellular physiology. However, the precise mechanisms underlying mitophagy remain largely unknown. Prior studies on mitophagy focused on the events in the mitochondrial outer membrane. PHB2 (prohibitin 2), which is a highly conserved membrane scaffold protein, was recently identified as a novel inner membrane mitophagy receptor that mediates mitophagy. Here, we report a new signaling pathway for PHB2-mediated mitophagy. Upon mitochondrial membrane depolarization or misfolded protein aggregation, PHB2 depletion destabilizes PINK1 in the mitochondria, which blocks the mitochondrial recruitment of PRKN/Parkin, ubiquitin and OPTN (optineurin), leading to an inhibition of mitophagy. In addition, PHB2 overexpression directly induces PRKN recruitment to the mitochondria. Moreover, PHB2-mediated mitophagy is dependent on the mitochondrial inner membrane protease PARL, which interacts with PHB2 and is activated upon PHB2 depletion. Furthermore, PGAM5, which is processed by PARL, participates in PHB2-mediated PINK1 stabilization. Finally, a ligand of PHB proteins that we synthesized, called FL3, was found to strongly inhibit PHB2-mediated mitophagy and to effectively block cancer cell growth and energy production at nanomolar concentrations. Thus, our findings reveal that the PHB2-PARL-PGAM5-PINK1 axis is a novel pathway of PHB2-mediated mitophagy and that targeting PHB2 with the chemical compound FL3 is a promising strategy for cancer therapy.: AIFM1: apoptosis inducing factor mitochondria associated 1; ATP5F1A/ATP5A1: ATP synthase F1 subunit alpha; BAF: bafilomycin A; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chemical reagent carbonyl cyanide m-chlorophenyl hydrazine; FL3: flavaglines compound 3; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryo fibroblasts; MPP: mitochondrial-processing peptidase; MT-CO2/COX2: mitochondrially encoded cytochrome c oxidase II; MTS: mitochondrial targeting sequence; OA: oligomycin and antimycin A; OPTN: optineurin; OTC: ornithine carbamoyltransferase; PARL: presenilin associated rhomboid like; PBS: phosphate-buffered saline; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PHB: prohibitin; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Roc-A: rocaglamide A; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin beta class I.
Topics: HCT116 Cells; HeLa Cells; Humans; Ligands; Metalloproteases; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Mitophagy; Phosphoprotein Phosphatases; Prohibitins; Protein Binding; Protein Kinases; Protein Stability; Repressor Proteins; Signal Transduction; Ubiquitin-Protein Ligases
PubMed: 31177901
DOI: 10.1080/15548627.2019.1628520 -
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 -
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 -
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 -
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 -
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 -
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 -
Cell Nov 2019Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways...
Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.
Topics: Autoantigens; Cell Death; Cytosol; Electron Transport Complex I; Epistasis, Genetic; Ferroptosis; Genes, Modifier; Genome; Glutathione Peroxidase; Glycolysis; Humans; K562 Cells; Mitochondria; Oligomycins; Oxidation-Reduction; Oxidative Phosphorylation; Pentose Phosphate Pathway; Reactive Oxygen Species; Ribonucleoproteins; SS-B Antigen
PubMed: 31730859
DOI: 10.1016/j.cell.2019.10.032 -
Stem Cell Research & Therapy Jun 2020Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is considered a promising therapeutic approach for bone defect repair. However, during the...
BACKGROUND
Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is considered a promising therapeutic approach for bone defect repair. However, during the transplantation procedure, the functions and viability of BMSCs may be impaired due to extended durations of in vitro culture, aging, and disease conditions of patients. Inspired by spontaneous intercellular mitochondria transfer that naturally occurs within injured tissues to rescue cellular or tissue function, we investigated whether artificial mitochondria transfer into pre-transplant BMSCs in vitro could improve cellular function and enhance their therapeutic effects on bone defect repair in situ.
METHODS
Mitochondria were isolated from donor BMSCs and transferred into recipient BMSCs of the same batch and passage. Subsequently, changes in proliferative capacity and cell senescence were evaluated by live cell imaging, Cell Counting Kit-8 assay, cell cycle analysis, Ki67 staining, qPCR and Western blot analysis of c-Myc expression, and β-galactosidase staining. Migration ability was evaluated by the transwell migration assay, wound scratch healing, and cell motility tests. Alkaline phosphatase (ALP) staining, Alizarin Red staining, and combined with qPCR and Western blot analyses of Runx2 and BMP2 were performed to elucidate the effects of mitochondria transfer on the osteogenic potential of BMSCs in vitro. After that, in vivo experiments were performed by transplanting mitochondria-recipient BMSCs into a rat cranial critical-size bone defect model. Micro CT scanning and histological analysis were conducted at 4 and 8 weeks after transplantation to evaluate osteogenesis in situ. Finally, in order to establish the correlation between cellular behavioral changes and aerobic metabolism, OXPHOS (oxidative phosphorylation) and ATP production were assessed and inhibition of aerobic respiration by oligomycin was performed.
RESULTS
Mitochondria-recipient BMSCs exhibited significantly enhanced proliferation and migration, and increased osteogenesis upon osteogenic induction. The in vivo results showed more new bone formation after transplantation of mitochondria-recipient BMSCs in situ. Increased OXPHOS activity and ATP production were observed, which upon inhibition by oligomycin attenuated the enhancement of proliferation, migration, and osteogenic differentiation induced by mitochondria transfer.
CONCLUSIONS
Mitochondria transfer is a feasible technique to enhance BMSC function in vitro and promote bone defect repair in situ through the upregulation of aerobic metabolism. The results indicated that mitochondria transfer may be a novel promising technique for optimizing stem cell therapeutic function.
Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Cells, Cultured; Humans; Mesenchymal Stem Cells; Mitochondria; Osteogenesis; Rats
PubMed: 32586355
DOI: 10.1186/s13287-020-01704-9