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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 -
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 -
Data in Brief Aug 2023The functional diversity of neurons is specified through their proteome resulting in elaborate and tightly regulated protein interaction networks and signalling that...
The functional diversity of neurons is specified through their proteome resulting in elaborate and tightly regulated protein interaction networks and signalling that regulates neuronal processes. Dysregulation of these dynamic networks in development or in adulthood lead to neurodevelopmental or neurological disorders respectively. Over the past few decades, mass spectrometry has become a powerful tool for quantifying and resolving any proteome, including complex tissues such as the brain proteome, with technological advances leading to higher levels of resolution and throughput than traditional biochemical techniques. In this article, we provide a proteomic reference dataset that has been generated to identify proteins and quantify their level of expression in primary mouse cortical neurons. It represents a summary analysis of previously published data in (Antico et al., 2021). Mouse cortical neurons were isolated from E16.5 C57Bl/6J mice and cultured for 21 (DIV). We employed the mitochondrial uncouplers AntimycinA/Oligomycin (AO) to induce mitochondrial depolarisation that is a well-established paradigm to assess mitophagic signalling. Total lysates from mouse primary cortical neurons were subjected to label-free quantitative proteomic analysis using both data dependent acquisition (DDA) and data independent acquisition (DIA) modes. DDA proteomic analysis identified a total dataset of 9367 proteins in mouse cortical neurons and absolute abundance of proteins was calculated as copy numbers per cell. DDA dataset was also processed to generate a reference spectral library to fit in and quantify MS spectra generated in DIA mode. Quantitative DIA analysis identified more than 6000 protein groups and statistical comparison of the two analysed groups (untreated and AO-treated) revealed that the neuronal proteome was largely unchanged post mitochondrial depolarisation for 5 hours. To our knowledge, these files represent the most comprehensive DDA and DIA reference datasets of fully functional maturated mouse primary cortical neurons and serve as a valuable resource for further investigating the role of specific proteins involved in neurobiology and neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Autism Spectrum Disorders (ASD).
PubMed: 37456110
DOI: 10.1016/j.dib.2023.109336 -
Molecules (Basel, Switzerland) Aug 2023Polyphenols have attracted attention in the fight against antibiotic-resistant bacteria, as they show antibacterial action. Considering that polyphenols inhibit FF-ATP...
Cirsiliol and Quercetin Inhibit ATP Synthesis and Decrease the Energy Balance in Methicillin-Resistant (MRSA) and Methicillin-Resistant (MRSE) Strains Isolated from Patients.
Polyphenols have attracted attention in the fight against antibiotic-resistant bacteria, as they show antibacterial action. Considering that polyphenols inhibit FF-ATP synthase (ATP synthase) and that bacteria need a constant energy production to maintain their homeostasis, we evaluated the effect of two flavones, cirsiliol (tri-hy-droxy-6,7-dimethoxyflavone) and quercetin (3,3,4,5,7-pentahydroxyflavone), on energy production and intracellular ATP content in a methicillin-resistant (MRSA) strain and a methicillin-resistant (MRSE) strain isolated from patients, comparing the results to those obtained by treating the bacteria with oligomycin, a specific ATP synthase F moiety inhibitor. Real-time quantitative ATP synthesis and total ATP content of permeabilized Gram-positive bacteria were assayed by luminometry. The results showed that cirsiliol and quercetin inhibited ATP synthase and decreased the intracellular ATP levels in both strains, although the effect was higher in MRSE. In addition, while cirsiliol and quercetin acted immediately after the treatment, oligomycin inhibited ATP synthesis only after 30 min of incubation, suggesting that the different responses may depend on the different permeability of the bacterial wall to the three molecules. Thus, cirsiliol and quercetin could be considered potential additions to antibiotics due to their ability to target ATP synthase, against which bacteria cannot develop resistance.
Topics: Humans; Methicillin-Resistant Staphylococcus aureus; Quercetin; Staphylococcus epidermidis; Methicillin Resistance; Flavones; Polyphenols; Adenosine Triphosphate; Anti-Bacterial Agents
PubMed: 37687012
DOI: 10.3390/molecules28176183 -
Biochimica Et Biophysica Acta.... Feb 2024Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear....
Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear. We have used N2a mouse neuroblastoma cells and primary cultures of mouse neurons and 1-methyl-4-phenylpyridinium (MPP), a known mitochondrial complex I inhibitor and the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), known to trigger a cascade of events associated with PD neuropathological features. Simultaneously, we utilized other mitochondrial toxins, including antimycin A, oligomycin, and carbonyl cyanide chlorophenylhydrazone. MPP treatment resulted in elevated levels of total cholesterol and in a Niemann Pick type C1 (NPC1)-like phenotype characterized by accumulation of cholesterol in lysosomes. Interestingly, NPC1 mRNA levels were specifically reduced by MPP. The decrease in NPC1 levels was also seen in midbrain and striatum from MPTP-treated mice and in primary cultures of neurons treated with MPP. Together with the MPP-dependent increase in intracellular cholesterol levels in N2a cells, we observed an increase in 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and a concomitant increase in the phosphorylated levels of mammalian target of rapamycin (mTOR). NPC1 knockout delayed cell death induced by acute mitochondrial damage, suggesting that transient cholesterol accumulation in lysosomes could be a protective mechanism against MPTP/MPP insult. Interestingly, we observed a negative correlation between NPC1 protein levels and disease stage, in human PD brain samples. In summary, MPP decreases NPC1 levels, elevates lysosomal cholesterol accumulation and alters mTOR signaling, adding to the existing notion that PD may rise from alterations in mitochondrial-lysosomal communication.
Topics: Animals; Humans; Mice; Cholesterol; Mammals; Mechanistic Target of Rapamycin Complex 1; Niemann-Pick C1 Protein; Parkinson Disease; Phenotype; TOR Serine-Threonine Kinases
PubMed: 38061599
DOI: 10.1016/j.bbadis.2023.166980 -
Neural Regeneration Research Jan 2024Mitochondrial dysfunction is a significant pathological alteration that occurs in Parkinson's disease (PD), and the Thr61Ile (T61I) mutation in coiled-coil helix...
Mitochondrial dysfunction is a significant pathological alteration that occurs in Parkinson's disease (PD), and the Thr61Ile (T61I) mutation in coiled-coil helix coiled-coil helix domain containing 2 (CHCHD2), a crucial mitochondrial protein, has been reported to cause Parkinson's disease. F1F0-ATPase participates in the synthesis of cellular adenosine triphosphate (ATP) and plays a central role in mitochondrial energy metabolism. However, the specific roles of wild-type (WT) CHCHD2 and T61I-mutant CHCHD2 in regulating F1F0-ATPase activity in Parkinson's disease, as well as whether CHCHD2 or CHCHD2 T61I affects mitochondrial function through regulating F1F0-ATPase activity, remain unclear. Therefore, in this study, we expressed WT CHCHD2 and T61I-mutant CHCHD2 in an MPP-induced SH-SY5Y cell model of PD. We found that CHCHD2 protected mitochondria from developing MPP-induced dysfunction. Under normal conditions, overexpression of WT CHCHD2 promoted F1F0-ATPase assembly, while T61I-mutant CHCHD2 appeared to have lost the ability to regulate F1F0-ATPase assembly. In addition, mass spectrometry and immunoprecipitation showed that there was an interaction between CHCHD2 and F1F0-ATPase. Three weeks after transfection with AAV-CHCHD2 T61I, we intraperitoneally injected 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into mice to establish an animal model of chronic Parkinson's disease and found that exogenous expression of the mutant protein worsened the behavioral deficits and dopaminergic neurodegeneration seen in this model. These findings suggest that WT CHCHD2 can alleviate mitochondrial dysfunction in PD by maintaining F1F0-ATPase structure and function.
PubMed: 37488867
DOI: 10.4103/1673-5374.378010 -
Journal of Agricultural and Food... Jan 2024Oligomycins are potent antifungal and antitumor agents. Mass spectrometry (MS)- and nuclear magnetic resonance (NMR)-based metabolomic fingerprinting analysis of...
Oligomycins are potent antifungal and antitumor agents. Mass spectrometry (MS)- and nuclear magnetic resonance (NMR)-based metabolomic fingerprinting analysis of marine-derived actinomycetes in our in-house library provided an oligomycin-producing strain, sp. FXY-T5. Chemical investigation led to the discovery of five new oligomycins, 24-lumooligomycin B (), 4-lumooligomycin B (), 6-lumooligomycin B (), 40-homooligomycin B (), and 15-hydroxy-oligomycin B (), together with seven biosynthetically related known derivatives. Their structures were assigned by MS, NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. The biosynthesis pathway of oligomycins was first proposed based on the analysis of a type I modular polyketide synthase (PKS) system and targeted gene disruption. As expected, the isolated oligomycins showed significant antiagricultural fungal pathogen activity and antiproliferative properties from which the possible structure-activity relationships were first suggested. More importantly, oligomycins induced significant G1-phase cell cycle arrest on cancer cells and significantly attenuated their Cyclin D1 and PCNA expression through a β-catenin signaling pathway.
Topics: Streptomyces; Oligomycins; Antineoplastic Agents; Structure-Activity Relationship; Antifungal Agents
PubMed: 38169320
DOI: 10.1021/acs.jafc.3c06307 -
Theriogenology Sep 2023Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the...
Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 10 mL in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 μM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 μM (CCCP), uncoupling agent; antimycin A 1 μg/mL (ANTI), complex III inhibitor; oligomycin 5 μM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O production and HO intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.
Topics: Male; Animals; Cattle; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Hydrogen Peroxide; Electrons; Semen; Sperm Motility; Spermatozoa; Mitochondria; Semen Preservation; Cryopreservation
PubMed: 37290146
DOI: 10.1016/j.theriogenology.2023.05.021 -
Anti-cancer Drugs Apr 2024The development of chemo-resistance in nasopharyngeal carcinoma (NPC) presents a significant therapeutic challenge, and its underlying mechanisms remain poorly...
The development of chemo-resistance in nasopharyngeal carcinoma (NPC) presents a significant therapeutic challenge, and its underlying mechanisms remain poorly understood. In our previous studies, we highlighted the association between isoprenylcysteine carboxylmethyltransferase (ICMT) and chemoresistance in NPC. In this current research, we revealed that both 5-FU and cisplatin-resistant NPC cells exhibited elevated mitochondrial function and increased expression of mitochondrial genes, independent of ICMT. Our investigations further showed that classic mitochondrial inhibitors, such as oligomycin, antimycin, and rotenone, were notably more effective in reducing viability in chemo-resistant NPC cells compared to parental cells. Moreover, we identified two antimicrobial drugs, tigecycline and atovaquone, recognized as mitochondrial inhibitors, as potent agents for decreasing chemo-resistant NPC cells by targeting mitochondrial respiration. Remarkably, tigecycline and atovaquone, administered at tolerable doses, inhibited chemo-resistant NPC growth in mouse models and extended overall survival rates. This work unveils the efficacy of mitochondrial inhibition as a promising strategy to overcome chemo-resistance in NPC. Additionally, our findings highlight the potential repurposing of clinically available drugs like tigecycline and atovaquone for treating NPC patients who develop chemoresistance.
Topics: Animals; Mice; Humans; Nasopharyngeal Carcinoma; Drug Resistance, Neoplasm; Atovaquone; Tigecycline; Cell Line, Tumor; Cisplatin; Mitochondria; Nasopharyngeal Neoplasms
PubMed: 38215016
DOI: 10.1097/CAD.0000000000001566 -
Oncology Research 2023Myc belongs to a family of proto-oncogenes that encode transcription factors. The overexpression of c-Myc causes many types of cancers. Recently, we established a system...
Myc belongs to a family of proto-oncogenes that encode transcription factors. The overexpression of c-Myc causes many types of cancers. Recently, we established a system for screening c-Myc inhibitors and identified antimycin A by screening the RIKEN NPDepo chemical library. The specific mechanism of promoting tumor cell metastasis by high c-Myc expression remains to be explained. In this study, we screened approximately 5,600 microbial extracts using this system and identified a broth prepared from sp. RK19-A0402 strongly inhibits c-Myc transcriptional activity. After purification of the hit broth, we identified compounds closely related to the aglycone of cytovaricin and had a structure similar to that of oligomycin A. Similar to oligomycin A, the hit compounds inhibited mitochondrial complex V. The mitochondria dysfunction caused by the compounds induced the production of reactive oxygen species (ROS), and the ROS activated GSK3α/β that phosphorylated c-Myc for ubiquitination. This study provides a successful screening strategy for identifying natural products as potential c-Myc inhibitors as potential anticancer agents.
Topics: Humans; Ubiquitin; Reactive Oxygen Species; Proto-Oncogene Proteins c-myc; Oligomycins
PubMed: 37547761
DOI: 10.32604/or.2023.030248