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Communications Biology Mar 2024Mitochondrial stress inducers such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and oligomycin trigger the DELE1-HRI branch of the integrated stress response...
Mitochondrial stress inducers such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and oligomycin trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged DELE1 showed that these stresses induced the cleavage of DELE1 to DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non-depolarizing MPIS. Surprisingly, while the mitochondrial protease OMA1 was crucial for DELE1 cleavage in HeLa cells, it was dispensable in HEK293T cells, suggesting that multiple proteases may be involved in DELE1 cleavage. In support, we identified a role for the mitochondrial protease, HtrA2, in mediating DELE1 cleavage into DELE1-VS, and showed that a Parkinson's disease (PD)-associated HtrA2 mutant displayed reduced DELE1 processing ability, suggesting a novel mechanism linking PD pathogenesis to mitochondrial stress. Our data further suggest that DELE1 is likely cleaved into DELE1-S in the cytosol, while the DELE1-VS form might be generated during halted translocation into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies a novel role for HtrA2 in DELE1 processing.
Topics: Humans; Cytosol; HEK293 Cells; HeLa Cells; Mitochondria; Mitochondrial Proteins
PubMed: 38555279
DOI: 10.1038/s42003-024-06107-7 -
Zhong Nan Da Xue Xue Bao. Yi Xue Ban =... Feb 2021Radiotherapy is one of the main therapies for colorectal cancer, but radioresistance often leads to radiotherapy failure. To improve the radioresistance, we explore the...
OBJECTIVES
Radiotherapy is one of the main therapies for colorectal cancer, but radioresistance often leads to radiotherapy failure. To improve the radioresistance, we explore the effect of oligomycin A, the H-ATP synthase inhibitor, on the sensitivity of HT29 colorectal cancer cells to irradiation and its underlying mechanisms.
METHODS
The effects of different concentrations of oligomycin A on the survival rate and glycolysis of HT29 colorectal cancer cells at different time points were investigated via MTT and glycolysis assay. siRNA-PFK1 was synthesized in vitro and transfected into HT29 cells. The effects of oligomycin A on radiosensitivity of HT29 colorectal cancer cells were measured via MTT and colony formation assay. Western blotting was used to detect the effect of oligomycin A on the expression of glycolytic enzyme PFK1. We compared difference between the effects of siRNA-PFK1 group and oligomycin A combined with siRNA-PFK1 group on cell survival and glycolysis. After 4 Gy X-ray irradiation, the effects of cell survival and glycolysis between the siRNA-PFK1 group and the oligomycin A combined with siRNA-PFK1 group were compared.
RESULTS
Compared with the 0 μmol/L oligomycin A group, the cell survival rate of HT29 cells treated with 4 μmol/L oligomycin A was significantly increased (<0.05), and the glucose uptake, the lactic acid, and the ATP production were also significantly increased (all <0.01). After X-ray irradiation at different doses (0, 2, 4, 6, and 8 Gy), the colony formation rate and cell survival rate of the 4 μmol/L oligomycin A treated group were significantly higher than those in the 0 μmol/L oligomycin A group (both <0.01). The sensitization enhancement ratio of oligomycin A on HT29 colorectal cancer cells was 0.4886. The expression of PFK1 in the 4 μmol/L oligomycin A group was significantly higher than that in the 0 μmol/L oligomycin A group (<0.001). The glycolysis level, colony formation rate, and cell survival rate of the siRNA-PFK1 HT29 cells group were significantly lower than those in the 0 μmol/L oligomycin A group (all <0.05), while the results in the 4 μmol/L oligomycin A combined with siRNA-PFK1 group were significantly higher than those in the siRNA-PFK1 group (all <0.001). After 4 Gy X-ray irradiation, the colony formation rate and cell survival rate in the siRNA-PFK1 group were decreased compared with those in the irradiation group (<0.01 or <0.001), while the results of the 4 μmol/L oligomycin A combined with siRNA-PFK1 group were significantly higher than those in the siRNA-PFK1 group (both <0.001).
CONCLUSIONS
Oligomycin A can promote the radioresistance of HT29 colorectal cancer cells, which may be related to up-regulation of the PFK1 expression and increase of cell glycolysis.
Topics: Cell Line, Tumor; Colorectal Neoplasms; HT29 Cells; Humans; Oligomycins; Radiation Tolerance
PubMed: 33678646
DOI: 10.11817/j.issn.1672-7347.2021.200063 -
Cells Feb 2023Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic...
Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.
Topics: Humans; Cytosol; HEK293 Cells; Adenosine Diphosphate; Adenosine Triphosphate; Atractyloside; Oligomycins; Stress, Physiological
PubMed: 36899830
DOI: 10.3390/cells12050695 -
Free Radical Biology & Medicine Mar 2024Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization...
Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart.
Topics: Adult; Mice; Humans; Animals; Myocytes, Cardiac; Reactive Oxygen Species; Cell Survival; Electron Transport Complex III; Antioxidants; Adenosine Triphosphate; Mitochondrial Diseases; Mammals
PubMed: 38266827
DOI: 10.1016/j.freeradbiomed.2024.01.034 -
Current Medicinal Chemistry 2022The major hurdles for successful cancer treatment are drug resistance and invasiveness developed by breast cancer stem cells (BCSC).
BACKGROUND
The major hurdles for successful cancer treatment are drug resistance and invasiveness developed by breast cancer stem cells (BCSC).
OBJECTIVE
As these two processes are highly energy-dependent, the identification of the main ATP supplier required for stem cell viability may result advantageous in the design of new therapeutic strategies to deter malignant carcinomas.
METHODS
The energy metabolism (glycolysis and oxidative phosphorylation, OxPhos) was systematically analyzed by assessing relevant protein contents, enzyme activities, and pathway fluxes in BCSC. Once identified as the main ATP supplier, selective energy inhibitors and canonical breast cancer drugs were used to block stem cell viability and metastatic properties.
RESULTS
OxPhos and glycolytic protein contents, as well as HK and LDH activities were several times higher in BCSC than in their parental line, MCF-7 cells. However, CS, GDH, COX activities, and both energy metabolism pathway fluxes were significantly lower (38-86%) in BCSC than in MCF-7 cells. OxPhos was the main ATP provider (>85%) in BCSC. Accordingly, oligomycin (a specific and potent canonical OxPhos inhibitor) and other non-canonical drugs with inhibitory effect on OxPhos (celecoxib, dimethylcelecoxib) significantly decreased BCSC viability, levels of epithelial-mesenchymal transition proteins, invasiveness, and induced ROS over-production, with IC values ranging from 1 to 20 μM in 24 h treatment. In contrast, glycolytic inhibitors (gossypol, iodoacetic acid, 3-bromopyruvate, 2-deoxyglucose) and canonical chemotherapeutic drugs (paclitaxel, doxorubicin, cisplatin) were much less effective against BCSC viability (IC> 100 μM).
CONCLUSION
These results indicated that the use of some NSAIDs may be a promising alternative therapeutic strategy to target BCSC.
Topics: Adenosine Triphosphate; Breast Neoplasms; Celecoxib; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Humans; Neoplastic Stem Cells; Oxidative Phosphorylation
PubMed: 34636290
DOI: 10.2174/0929867328666211005124015 -
Medical Oncology (Northwood, London,... Jul 2020Cancer cells alter their metabolism by switching from glycolysis to oxidative phosphorylation (OXPHOS), regardless of oxygen availability. Metabolism may be a molecular...
Cancer cells alter their metabolism by switching from glycolysis to oxidative phosphorylation (OXPHOS), regardless of oxygen availability. Metabolism may be a molecular target in acute myeloid leukemia (AML), where mutations in metabolic genes have been described. This study evaluated glycolysis and OXPHOS as therapeutic targets. The sensitivity to 2-deoxy-D-glucose (2-DG; glycolysis inhibitor) and oligomycin (OXPHOS inhibitor) was tested in six AML cell lines (HEL, HL-60, K-562, KG-1, NB-4, THP-1). These cells were characterized for IDH1/2 exon 4 mutations, reactive oxygen species, and mitochondrial membrane potential. Metabolic activity was assessed by resazurin assay, whereas cell death and cell cycle were assessed by flow cytometry. Glucose uptake and metabolism-related gene expression were analyzed by F-FDG and RT-PCR/qPCR, respectively. No IDH1/2 exon 4 mutations were detected. HEL cells had the highest F-FDG uptake and peroxides/superoxide anion levels, whereas THP-1 showed the lowest. 2-DG reduced metabolic activity in all cell lines with HEL, KG-1, and NB-4 being the most sensitive cells. Oligomycin decreased metabolic activity in a cell line-dependent manner, the THP-1 resistant and HL-60 being the most sensitive. Both inhibitors induced apoptosis and cell cycle arrest in a cell line- and compound-dependent manner. 2-DG decreased F-FDG uptake in HEL, HL-60, KG-1, and NB-4, while oligomycin increased the uptake in K-562. Metabolism gene expression had different responses to treatments. In conclusion, HEL and KG-1 show to be more glycolytic, whereas HL-60 was more OXPHOS dependent. Results suggest that AML cells reprogram their metabolism to overcome OXPHOS inhibition suggesting that glycolysis may be a better therapeutic target.
Topics: Anti-Bacterial Agents; Antimetabolites; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Deoxyglucose; Glucose; Glycolysis; Humans; Leukemia, Myeloid, Acute; Oligomycins; Oxidative Phosphorylation
PubMed: 32725458
DOI: 10.1007/s12032-020-01394-6 -
Theriogenology Mar 2020In this study boar sperm mitochondrial activity was studied and deepened in order to delineate the main metabolic strategies used by boar sperm to obtain energy and to...
In this study boar sperm mitochondrial activity was studied and deepened in order to delineate the main metabolic strategies used by boar sperm to obtain energy and to link them to sperm function. Boar spermatozoa were collected, diluted at 30 × 10 spz/mL and incubated for 1 h with: Rotenone (ROT), complex I inhibitor, Dimethyl-malonate (DMM), complex II inhibitor, antimycin A (ANTI), complex III inhibitor, oligomycin (OLIGO), ATP synthase inhibitor, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), uncoupling agent, 2-deoxy-glucose (2DG), glucose agonist, and Dimethyl sulphoxide (DMSO) as control vehicle. Viability and mitochondrial membrane potential (Sybr14/PI/JC1 staining) and sperm motility (using CASA system) were assayed after incubation. ROT, ANTI, OLIGO and CCCP significantly reduced total and progressive motility as well as cell velocities; ANTI and CCCP depressed mitochondrial membrane potential but did not affect cell viability. Cluster analysis of kinematic parameters showed some interesting features of sperm subpopulations: ANTI and CCCP caused a shift in sperm subpopulation towards "slow non progressive" cells, OLIGO and ROT caused a shift towards "average" and "slow non progressive" cells, while DMM and 2DG increased the "fast progressive" cells subpopulation. Sperm mitochondrial respiration and substrate oxidation, assayed polographically and spectrofluorimetrically, respectively pointed out a high ATP turnover and a low spare respiratory capacity, mainly linked to the NADH-O oxidase activity. Therefore, boar spermatozoa heavily rely on mitochondrial oxidative phosphorylation, and especially on Complex I activity, to produce ATP and fuel motility.
Topics: Animals; Cell Survival; Male; Membrane Potential, Mitochondrial; Mitochondria; Oxidation-Reduction; Oxygen Consumption; Principal Component Analysis; Sperm Motility; Spermatozoa; Swine
PubMed: 31927418
DOI: 10.1016/j.theriogenology.2020.01.004 -
Experimental Cell Research Apr 2021We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis...
We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis elegans. Herein, we discovered that the ciliary localization of ATP synthase is evolutionarily conserved in mammals. We showed that the ATP synthase subunit F1β is colocalized with the cilia marker acetylated α-tubulin in both mammalian renal epithelial cells (MDCK) and normal mouse cholangiocytes (NMCs). Treatment with ATP synthase inhibitor oligomycin impaired ciliogenesis in MDCK cells, and F1β was co-immunoprecipitated with PKD2 in mammalian cells. Our study provides evidence for the evolutionarily conserved localization of ATP synthase in cilia from worm to mammals. Defects in ATP synthase can lead to ciliary dysfunction, which may be a potential mechanism of polycystic kidney disease.
Topics: ATP Synthetase Complexes; Adenosine Triphosphate; Animals; Caenorhabditis elegans; Cilia; Dogs; Kinesins; Madin Darby Canine Kidney Cells; Mammals; Mice; Mitochondrial Proton-Translocating ATPases; Molecular Chaperones; Oligomycins; Polycystic Kidney Diseases; Protein Processing, Post-Translational; TRPP Cation Channels
PubMed: 33639177
DOI: 10.1016/j.yexcr.2021.112520 -
Journal of Visualized Experiments : JoVE Feb 2022Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as...
Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate. Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers, DNA content and type. The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.
Topics: Cell Culture Techniques; Cell Differentiation; Energy Metabolism; Mitochondria; Spheroids, Cellular
PubMed: 35188130
DOI: 10.3791/63346 -
Cellular Signalling Apr 2022The mitochondrial unfolded protein response (UPR) is an adaptive transcriptional response involving the activation of proteases, chaperones, and antioxidant enzymes and...
The mitochondrial unfolded protein response (UPR) is an adaptive transcriptional response involving the activation of proteases, chaperones, and antioxidant enzymes and serves to degrade abnormal or unfolded proteins and restore mitochondrial function. Although the cardioprotective action of the UPR has been verified in myocardial ischemia/reperfusion (I/R) injuries, the upstream signals involved remain unclear. Here, we explored the regulatory mechanisms underlying UPR in the reperfused mouse heart. UPR was slightly activated by I/R injury. UPR activation (using oligomycin) and inhibition (with the protease inhibitor AEBSF) respectively alleviated and augmented the reperfusion-mediated myocardial damage. Gene expression analysis demonstrated that oxidative stress was partly inhibited by UPR through upregulation of mitochondria-localized, not cytoplasmic, antioxidant enzymes. Contributing to cardiomyocyte survival under I/R, the transcription of pro-apoptotic proteins Bcl2 and c-IAP was also stimulated by UPR. Moreover, UPR upregulated mitochondrial fusion-related, but not fission-related, genes and stimulated the expression of mitochondrial biogenesis markers in reperfused hearts. Finally, we found that FUN14 domain containing 1 (FUNDC1)-mediated mitophagy induces the mitochondrial DNA decrease, triggering UPR. These results demonstrate that FUNDC1 functions upstream of the UPR to maintain mitochondrial quality control during myocardial I/R injury.
Topics: Animals; Ischemia; Membrane Proteins; Mice; Mitochondria; Mitochondrial Proteins; Myocardial Reperfusion Injury; Unfolded Protein Response
PubMed: 35051611
DOI: 10.1016/j.cellsig.2022.110249