TRAP1 inhibits MIC60 ubiquitination to mitigate the injury of cardiomyocytes and protect mitochondria in extracellular acidosis.
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ABSTRACT:
SUBMITTER: Zhang L
PROVIDER: S-EPMC8671480 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitochondria are tubular double-membrane organelles essential for eukaryotic life. They form extended networks and exhibit an intricate inner membrane architecture. The MICOS (mitochondrial contact site and cristae organizing system) complex, crucial for proper architecture of the mitochondrial inner membrane, is localized primarily at crista junctions. Harnessing superresolution fluorescence microscopy, we demonstrate that Mic60, a subunit of the MICOS complex, as well as several of its interaction partners are arranged into intricate patterns in human and yeast mitochondria, suggesting an ordered distribution of the crista junctions. We show that Mic60 forms clusters that are preferentially localized in the inner membrane at two opposing sides of the mitochondrial tubules so that they form extended opposing distribution bands. These Mic60 distribution bands can be twisted, resulting in a helical arrangement. Focused ion beam milling-scanning electron microscopy showed that in yeast the twisting of the opposing distribution bands is echoed by the folding of the inner membrane. We show that establishment of the Mic60 distribution bands is largely independent of the cristae morphology. We suggest that Mic60 is part of an extended multiprotein interaction network that scaffolds mitochondria.
| S-EPMC6525524 | biostudies-literature
Project description:A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis.Acidosis increased both glutaminolysis and fatty acid ?-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes.Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.
| S-EPMC4178214 | biostudies-other
Project description:Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (?-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1? and CA IX and/or altered CA IX function.The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1? and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively.Carnosine increased the expression levels of HIF-1? and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.
| S-EPMC4061103 | biostudies-literature
Project description:Platelets are classically known for their roles in bleeding control and occlusive thrombus formation causing ischaemic tissue damage. Recently non-classical roles for platelets have been described, many of which may be mediated by the heterogeneous cargo that platelets secrete from granular stores upon activation. Using an in vitro model of ischaemic injury to ventricular cardiomyocytes, we observed that platelets, through secreted factors, delayed the rate of cardiomyocyte death during ischaemia. This protective effect appeared independent of platelet dense granule cargo, but required α-granule components stromal cell-derived factor (SDF)-1α and transforming growth factor (TGF) β1. Protein kinase C (PKC) activity within cardiomyocytes was responsible for mediating the protective signals initiated by the released platelet cargo. Importantly, pretreating platelets with a P2Y12 antagonist, but not the cyclooxygenase inhibitor aspirin, substantially attenuated this protective effect. These findings therefore reveal a paradoxically protective role for platelet activation during cardiac ischaemia and could have important implications for the use of anti-platelet therapeutics in the management of myocardial infarction.
| S-EPMC6241850 | biostudies-literature
Project description:BackgroundAnthracycline-induced cardiomyopathy (AIC) is a significant source of morbidity and mortality in cancer survivors. The role of mesenchymal stem cells (MSCs) in treating AIC was evaluated in the SENECA trial, a Phase 1 National Heart, Lung, and Blood Institute-sponsored study, but the mechanisms underpinning efficacy in human tissue need clarification.ObjectivesThe purpose of this study was to perform an in vitro clinical trial evaluating the efficacy and putative mechanisms of SENECA trial-specific MSCs in treating doxorubicin (DOX) injury, using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iCMs) generated from SENECA patients.MethodsPatient-specific iCMs were injured with 1 μmol/L DOX for 24 hours, treated with extracellular vesicles (EVs) from MSCs by either coculture or direct incubation and then assessed for viability and markers of improved cellular physiology. MSC-derived EVs were separated into large extracellular vesicles (L-EVs) (>200 nm) and small EVs (<220nm) using a novel filtration system.ResultsiCMs cocultured with MSCs in a transwell system demonstrated improved iCM viability and attenuated apoptosis. L-EVs but not small EVs recapitulated this therapeutic effect. L-EVs were found to be enriched in mitochondria, which were shown to be taken up by iCMs. iCMs treated with L-EVs demonstrated improved contractility, reactive oxygen species production, ATP production, and mitochondrial biogenesis. Inhibiting L-EV mitochondrial function with 1-methyl-4-phenylpyridinium attenuated efficacy.ConclusionsL-EV-mediated mitochondrial transfer mitigates DOX injury in patient-specific iCMs. Although SENECA was not designed to test MSC efficacy, consistent tendencies toward a positive effect were observed across endpoints. Our results suggest a mechanism by which MSCs may improve cardiovascular performance in AIC independent of regeneration, which could inform future trial design evaluating the therapeutic potential of MSCs.
| S-EPMC8463733 | biostudies-literature
Project description:IntroductionRegenerative medicine provides promising approaches for treating chronic liver diseases. Previous studies indicate that decellularized liver architecture is damaged by invading non-hepatic inflammatory cells. This study aimed to use anti-inflammatory and regenerative potency of bone marrow-derived mesenchymal stem cells (BM-MSC) and prednisolone for reducing fibrosis and balancing inflammatory cell migration into the decellularized liver scaffold.Material and methodThe liver was decellularized by perfusing Sodium Lauryl Ether Sulfate (SLES), and nuclei depletion and extracellular matrix (ECM) retention were confirmed by DNA quantification, histochemical, and immunohistochemical assessments. Scaffolds were loaded with BM-MSCs, prednisolone, or a combination of both, implanted at the anatomical place in the rat partial hepatectomized and followed up for 2 and 4 weeks.ResultsLabeled-MSCs were traced in the transplanted scaffolds; however, they did not migrate into the intact liver. Immunohistochemistry showed that the hepatoblasts, cholangiocytes, stellate, and oval cells invaded into all the scaffolds. Bile ducts were more abundant in the border of the scaffolds and intact liver. Stereological assessments showed a significant reduction in the number of lymphocytes and neutrophils in prednisolone-loaded scaffolds. The regeneration process and angiogenesis were significantly higher in the group treated with cell/prednisolone-loaded bioscaffolds. Collagen fibers were significantly reduced in the scaffolds pre-treated with cell/prednisolone, prednisolone, or BM-MSCs, compared to the control group.ConclusionLoading prednisolone into the scaffolds can be a worthy approach to restrict inflammation after transplantation. Although pre-loading of the scaffolds with a combination of cells/prednisolone could not alleviate inflammation, it played an important role in regeneration and angiogenesis.
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Project description:Numerous studies have established the involvement of lysosomal and mitochondrial dysfunction in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson diseases. Building on our previous studies of the neurodegenerative lysosomal lipidosis Niemann-Pick C1 (NPC1), we have unexpectedly discovered that activation of the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) leads to the correction of the lysosomal storage phenotype in patient cells from multiple lysosomal storage disorders including NPC1. Using small compound activators specific for TRAP1, we find that activation of this chaperone leads to a generalized restoration of lysosomal and mitochondrial health. Mechanistically, we show that this process includes inhibition of oxidative phosphorylation and reduction of oxidative stress, which results in activation of AMPK and ultimately stimulates lysosome recycling. Thus, TRAP1 participates in lysosomal-mitochondrial crosstalk to maintain cellular homeostasis and could represent a potential therapeutic target for multiple disorders.
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Project description:Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of ?7?1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of ?1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin ?7?1D exposed to I/R had a substantial reduction in infarct size compared with that of ?5?1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that ?7?1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that ?1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165-175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that ?7?1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury.
| S-EPMC3784521 | biostudies-literature
Project description:TcpC is a multifunctional virulence factor of uropathogenic E. coli (UPEC). Neutrophil extracellular trap formation (NETosis) is a crucial anti-infection mechanism of neutrophils. Here we show the influence of TcpC on NETosis and related mechanisms. We show NETosis in the context of a pyelonephritis mouse model induced by TcpC-secreting wild-type E. coli CFT073 (CFT073wt) and LPS-induced in vitro NETosis with CFT073wt or recombinant TcpC (rTcpC)-treated neutrophils are inhibited. rTcpC enters neutrophils through caveolin-mediated endocytosis and inhibits LPS-induced production of ROS, proinflammatory cytokines and protein but not mRNA levels of peptidylarginine deiminase 4 (PAD4). rTcpC treatment enhances PAD4 ubiquitination and accumulation in proteasomes. Moreover, in vitro ubiquitination kit analyses show that TcpC is a PAD4-targetd E3 ubiquitin-ligase. These data suggest that TcpC inhibits NETosis primarily by serving as an E3 ligase that promotes degradation of PAD4. Our findings provide a novel mechanism underlying TcpC-mediated innate immune evasion.
| S-EPMC8190435 | biostudies-literature
Project description:Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.
| S-EPMC9974867 | biostudies-literature