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The Journal of Biological Chemistry Dec 2023Mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly...
Mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly interact in Saccharomyces cerevisiae to facilitate membrane scission. However, it remains unclear if a direct interaction is conserved in higher eukaryotes as other Drp1 recruiters, not present in yeast, are known. Using NMR, differential scanning fluorimetry, and microscale thermophoresis, we determined that human Fis1 directly interacts with human Drp1 (K = 12-68 μM), and appears to prevent Drp1 assembly, but not GTP hydrolysis. Similar to yeast, the Fis1-Drp1 interaction appears governed by two structural features of Fis1: its N-terminal arm and a conserved surface. Alanine scanning mutagenesis of the arm identified both loss-of-function and gain-of-function alleles with mitochondrial morphologies ranging from highly elongated (N6A) to highly fragmented (E7A), demonstrating a profound ability of Fis1 to govern morphology in human cells. An integrated analysis identified a conserved Fis1 residue, Y76, that upon substitution to alanine, but not phenylalanine, also caused highly fragmented mitochondria. The similar phenotypic effects of the E7A and Y76A substitutions, along with NMR data, support that intramolecular interactions occur between the arm and a conserved surface on Fis1 to promote Drp1-mediated fission as in S. cerevisiae. These findings indicate that some aspects of Drp1-mediated fission in humans derive from direct Fis1-Drp1 interactions that are conserved across eukaryotes.
Topics: Humans; Alanine; Dynamins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Saccharomyces cerevisiae
PubMed: 37866629
DOI: 10.1016/j.jbc.2023.105380 -
FASEB Journal : Official Publication of... Jan 2024Dynamin-related protein 1 (Drp1) is a cytosolic GTPase protein that when activated translocates to the mitochondria, meditating mitochondrial fission and increasing...
Dynamin-related protein 1 (Drp1) is a cytosolic GTPase protein that when activated translocates to the mitochondria, meditating mitochondrial fission and increasing reactive oxygen species (ROS) in cardiomyocytes. Drp1 has shown promise as a therapeutic target for reducing cardiac ischemia/reperfusion (IR) injury; however, the lack of specificity of some small molecule Drp1 inhibitors and the reliance on the use of Drp1 haploinsufficient hearts from older mice have left the role of Drp1 in IR in question. Here, we address these concerns using two approaches, using: (a) short-term (3 weeks), conditional, cardiomyocyte-specific, Drp1 knockout (KO) and (b) a novel, highly specific Drp1 GTPase inhibitor, Drpitor1a. Short-term Drp1 KO mice exhibited preserved exercise capacity and cardiac contractility, and their isolated cardiac mitochondria demonstrated increased mitochondrial complex 1 activity, respiratory coupling, and calcium retention capacity compared to controls. When exposed to IR injury in a Langendorff perfusion system, Drp1 KO hearts had preserved contractility, decreased reactive oxygen species (ROS), enhanced mitochondrial calcium capacity, and increased resistance to mitochondrial permeability transition pore (MPTP) opening. Pharmacological inhibition of Drp1 with Drpitor1a following ischemia, but before reperfusion, was as protective as Drp1 KO for cardiac function and mitochondrial calcium homeostasis. In contrast to the benefits of short-term Drp1 inhibition, prolonged Drp1 ablation (6 weeks) resulted in cardiomyopathy. Drp1 KO hearts were also associated with decreased ryanodine receptor 2 (RyR2) protein expression and pharmacological inhibition of the RyR2 receptor decreased ROS in post-IR hearts suggesting that changes in RyR2 may have a role in Drp1 KO mediated cardioprotection. We conclude that Drp1-mediated increases in myocardial ROS production and impairment of mitochondrial calcium handling are key mechanisms of IR injury. Short-term inhibition of Drp1 is a promising strategy to limit early myocardial IR injury which is relevant for the therapy of acute myocardial infarction, cardiac arrest, and heart transplantation.
Topics: Animals; Mice; Calcium; Dynamins; Homeostasis; Mitochondria, Heart; Mitochondrial Dynamics; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Reactive Oxygen Species; Ryanodine Receptor Calcium Release Channel
PubMed: 38133921
DOI: 10.1096/fj.202301040RR -
Acta Biomaterialia Oct 2023Although mitochondrial morphology and function are considered to be closely related to matrix stiffness-driven tumor progression, it remains poorly understood how...
Although mitochondrial morphology and function are considered to be closely related to matrix stiffness-driven tumor progression, it remains poorly understood how extracellular matrix (ECM) stiffness affects mitochondrial dynamics and mitophagy. Here, we found that soft substrate triggered calcium transport by increasing endoplasmic reticulum (ER) calcium release and mitochondrial (MITO) calcium uptake. ER-MITO calcium transport promoted the recruitment of dynamin-related protein 1 (Drp1) to mitochondria and phosphorylation at the serine 616 site, which induced mitochondrial fragmentation and Parkin/PINK1-mediated mitophagy. Furthermore, in vivo experiments demonstrated that soft ECM enhanced calcium levels in tumor tissue, Drp1 activity was required for soft ECM-induced mitochondrial dynamics impairment, and inhibition of Drp1 activity enhanced soft ECM-induced tumor necrosis. In conclusion, we revealed a new mechanism whereby ER-MITO calcium transport regulated mitochondrial dynamics and mitophagy through Drp1 translocation in response to soft substrates. These findings provide valuable insights into ECM stiffness as a potential target for antitumor therapy. STATEMENT OF SIGNIFICANCE: Here, we examined the relationship between substrate stiffness and mitochondrial dynamics by using polyacrylamide (PAA) substrates to simulate the stages of breast cancer or BAPN to reduce tumor tissue stiffness. The results elucidated that soft substrate triggered the recruitment of DRP1 and subsequent mitochondrial fission and mitophagy by ER-MITO calcium transport. Furthermore, mitophagy partly attenuated soft ECM-mediated tumor tissue necrosis and contributed to tumor survival in vivo. Our discoveries revealed the molecular mechanisms by which mechanical stimulation regulates mitochondrial dynamics, providing valuable insights into ECM stiffness as a target for anti-tumor approaches, which could be beneficial for both biomechanics research and clinical applications.
Topics: Humans; Female; Mitophagy; Mitochondrial Dynamics; Calcium; Dynamins; Endoplasmic Reticulum; Necrosis; Breast Neoplasms
PubMed: 37541606
DOI: 10.1016/j.actbio.2023.07.060 -
Autophagy Jun 2024RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS)...
RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS) generation, which we previously demonstrated as a trigger for autophagy-associated execution of mutant KRAS-expressing cancer cells. Here we report that drug (merodantoin; C1)-induced activation of mutant KRAS promotes phospho-AKT S473-dependent ROS-mediated S616 phosphorylation and mitochondrial localization of DNM1L/DRP1 (dynamin 1 like) and cleavage of the fusion-associated protein OPA1 (OPA1 mitochondrial dynamin like GTPase). Interestingly, accumulation of the outer mitochondrial membrane protein VDAC1 (voltage dependent anion channel 1) is observed in mutant KRAS-expressing cells upon exposure to C1. Conversely, silencing abolishes C1-induced mitophagy, and gene knockdown of either , rescues ROS-dependent VDAC1 accumulation and stability, thus suggesting an axis of mutant active KRAS-phospho-AKT S473-ROS-DNM1L-VDAC1 in mitochondrial morphology change and cancer cell execution. Importantly, we identified MTOR (mechanistic target of rapamycin kinsase) complex 2 (MTORC2) as the upstream mediator of AKT phosphorylation at S473 in our model. Pharmacological or genetic inhibition of MTORC2 abrogated C1-induced phosphorylation of AKT S473, ROS generation and mitophagy induction, as well as rescued tumor colony forming ability and migratory capacity. Finally, increase in thermal stability of KRAS, AKT and DNM1L were observed upon exposure to C1 only in mutant KRAS-expressing cells. Taken together, our work has unraveled a novel mechanism of selective targeting of mutant KRAS-expressing cancers via MTORC2-mediated AKT activation and ROS-dependent mitofission, which could have potential therapeutic implications given the relative lack of direct RAS-targeting strategies in cancer.: ACTB/ß-actin: actin beta; AKT: AKT serine/threonine kinase; C1/merodantoin: 1,3-dibutyl-2-thiooxo-imidazoldine-4,5-dione; CAT: catalase; CETSA: cellular thermal shift assay; CHX: cycloheximide; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HO: hydrogen peroxide; HSPA1A/HSP70-1: heat shock protein family A (Hsp70) member 1A; HSP90AA1/HSP90: heat shock protein 90 alpha family class A member 1; KRAS: KRAS proto-oncogene, GTPase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; LC3B-I: unlipidated form of LC3B; LC3B-II: phosphatidylethanolamine-conjugated form of LC3B; MAPKAP1/SIN1: MAPK associated protein 1; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MFI: mean fluorescence intensity; MiNA: Mitochondrial Network Analysis; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; O.: superoxide; OMA1: OMA1 zinc metallopeptidase; OPA1: OPA1 mitochondrial dynamin like GTPase; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR/raptor: regulatory associated protein of MTOR complex 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; VDAC1: voltage dependent anion channel 1; VDAC2: voltage dependent anion channel 2.
Topics: Humans; Reactive Oxygen Species; Proto-Oncogene Proteins c-akt; Mitophagy; Proto-Oncogene Proteins p21(ras); Colorectal Neoplasms; Mitochondria; Mechanistic Target of Rapamycin Complex 2; Mutation; Signal Transduction; Cell Line, Tumor; Phosphorylation
PubMed: 38261660
DOI: 10.1080/15548627.2024.2307224 -
Frontiers in Immunology 2023Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology,...
Macrophages play a critical role in the regulation of inflammation and tissue homeostasis. In addition to their vital functions for cell survival and physiology, mitochondria play a crucial role in innate immunity as a platform for the induction of inflammatory responses by regulating cell signaling and dynamics. Dynamin-related protein 1 (Drp1) plays a role in the induction of inflammatory responses and the subsequent development of various diseases. PGAM5 (phosphoglycerate mutase member 5) is a mitochondrial outer membrane phosphatase that dephosphorylates its substrate, Drp1. Previous studies showed that PGAM5 regulates the phosphorylation of Drp1 for the activation of NKT cells and T cells. However, it is not clear how PGAM5 regulates Drp1 activity for the induction of inflammation in macrophages. Here, we demonstrate that PGAM5 activity regulates the dephosphorylation of Drp1 in macrophages, leading to the induction of proinflammatory responses in macrophages. In TLR signaling, PGAM5 regulates the expression and production of inflammatory cytokines by regulating the activation of downstream signaling pathways, including the NF-κB and MAPK pathways. Upon LPS stimulation, PGAM5 interacts with Drp1 to form a complex, leading to the production of mtROS. Furthermore, PGAM5-Drp1 signaling promotes the polarization of macrophages toward a proinflammatory phenotype. Our study further demonstrates that PGAM5-Drp1 signaling promotes metabolic reprogramming by upregulating glycolysis and mitochondrial metabolism in macrophages. Altogether, PGAM5 signaling is a linker between alterations in Drp1-mediated mitochondrial dynamics and inflammatory responses in macrophages and may be a target for the treatment of inflammatory diseases.
Topics: Humans; Dynamins; Inflammation; Macrophages; Mitochondrial Proteins; Phosphoprotein Phosphatases; Signal Transduction; Animals
PubMed: 37771598
DOI: 10.3389/fimmu.2023.1243548 -
Science Signaling Aug 2023Fasting activates mTORC2 to stimulate mitochondrial fission and support mitochondrial respiration.
Fasting activates mTORC2 to stimulate mitochondrial fission and support mitochondrial respiration.
Topics: Dynamins; Mitochondrial Dynamics; Mitochondria; Fasting; Mitochondrial Proteins
PubMed: 37552768
DOI: 10.1126/scisignal.adk1008 -
ENeuro Aug 2023As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2...
As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2 (MFN2)/mitochondrial assembly regulatory factor (Marf), plays a role in mitochondrial fusion, mutations of which are implicated in age-related human diseases, including several neurodegenerative disorders. However, the regulation of MFN2/Marf-mediated mitochondrial fusion, as well as the pathologic mechanism of neurodegeneration, is not clearly understood. Here, we identified a novel interaction between MFN2/Marf and microtubule affinity-regulating kinase 4 (MARK4)/PAR-1. In the larval neuromuscular junction, muscle-specific overexpression of MFN2/Marf decreased the number of synaptic boutons, and the loss of MARK4/PAR-1 alleviated the synaptic defects of MFN2/Marf overexpression. Downregulation of MARK4/PAR-1 rescued the mitochondrial hyperfusion phenotype caused by MFN2/Marf overexpression in the muscles as well as in the cultured cells. In addition, knockdown of MARK4/PAR-1 rescued the respiratory dysfunction of mitochondria induced by MFN2/Marf overexpression in mammalian cells. Together, our results indicate that the interaction between MFN2/Marf and MARK4/PAR-1 is fine-tuned to maintain synaptic integrity and mitochondrial homeostasis, and its dysregulation may be implicated in neurologic pathogenesis.
Topics: Animals; Humans; Drosophila; Drosophila Proteins; GTP Phosphohydrolases; Mammals; Microtubules; Mitochondria; Mitochondrial Proteins; Protein Serine-Threonine Kinases; Synapses
PubMed: 37550059
DOI: 10.1523/ENEURO.0409-22.2023 -
The Science of the Total Environment Dec 2023As one of the major substitutes for bisphenol A (BPA), bisphenol F (BPF) has been widely used. Our previous study demonstrated that BPF exposure facilitates lipid...
As one of the major substitutes for bisphenol A (BPA), bisphenol F (BPF) has been widely used. Our previous study demonstrated that BPF exposure facilitates lipid droplet deposition in hepatic cells, contributing to nonalcoholic fatty liver disease (NAFLD)-like changes. However, the underlying mechanisms remain poorly understood. Here, with a metabolic cage system, we observed the perturbation of energy metabolism in mice treated with BPF. BPF obviously suppressed metabolic capacity, which manifested as decreased energy expenditure, low O consumption and CO levels in mice. Consistent with the in vivo results, a Seahorse XF Cell Mito Stress Test showed significant reductions in mitochondrial ATP production capacity, maximum respiratory capacity, and residual respiratory capacity after BPF treatment in an in vitro study. Electron microscopy revealed a striking increase in mitochondrial fission that was synchronous with excessive expression and activation of dynamin-related protein 1 (Drp1). Intriguingly, chemical inhibition of Drp1 by Mdivi-1 and/or silencing of Drp1 dramatically hampered mitochondrial fission and ameliorated BPF-induced lipid droplet deposition both in mouse liver and human hepatic cells. Mechanistically, mitochondrial dynamics imbalance played prominent roles in these processes, since suppression of Drp1 by chemical inhibition or knockdown substantially reversed BPF-induced mitochondrial fission and ameliorated the suppression of mitochondrial metabolism as well as excessive mitochondrial ROS, which was verified to be key to lipid droplet deposition. Collectively, the findings of the current study reveal previously unrecognized effects involving Drp1-mediated mitochondrial injury in BPF-induced lipid droplet deposition. Therefore, targeted intervention against mitochondrial dysfunction may be a promising therapeutic strategy for BPF-induced NAFLD-like changes.
Topics: Animals; Humans; Mice; Dynamins; Lipid Droplets; Mitochondrial Dynamics; Non-alcoholic Fatty Liver Disease
PubMed: 37683851
DOI: 10.1016/j.scitotenv.2023.166831 -
Cryobiology Dec 2023Acute extreme cold exposure impairs human health and even causes hypothermia which threatens human life. Liver, as a hub in metabolism and thermogenesis, is vital for...
Acute extreme cold exposure impairs human health and even causes hypothermia which threatens human life. Liver, as a hub in metabolism and thermogenesis, is vital for cold acclimatization. Although accumulating evidence has suggested that cold exposure can cause liver damage, the underlying mechanisms remain poorly understood. This study investigated the role and underlying mechanisms of ferroptosis in cold stress-induced liver damage. To evaluate the role of ferroptosis in cold stress-induced liver damage, rats were pretreated with ferroptosis inhibitor liproxstatin-1 (Lip-1) before exposed to -10 °C for 8 h. Core body temperature was recorded. The levels of ferroptosis-related indicators were examined with the corresponding assay kits or by western blotting. Hepatic pathological changes were analyzed by hematoxylin-eosin staining and ultrastructural observation. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured to assess liver function. Rats were also pretreated with p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or Dynamin-related protein 1 (Drp1) inhibitor Mdivi-1 to determine the underlying mechanisms. We found that Lip-1 inhibited ferroptosis, attenuated hepatic pathological damages and blocked the increased ALT and AST levels in cold-exposed rats. Moreover, Mdivi-1 inhibited mitochondrial fission and suppressed ferroptosis. Furthermore, SB203580 and Mdivi-1 administration alleviated cold stress-induced liver injury. Our results suggested that cold stress caused liver damage partially by inducing ferroptosis through the p38 MAPK/Drp1 pathway. These findings might provide an effective preventive and therapeutic target for cold stress-induced liver injury.
Topics: Rats; Humans; Animals; p38 Mitogen-Activated Protein Kinases; Cold-Shock Response; Chemical and Drug Induced Liver Injury, Chronic; Ferroptosis; Cryopreservation; Dynamins; Liver
PubMed: 37532122
DOI: 10.1016/j.cryobiol.2023.104563 -
Nature Communications Mar 2024NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in...
NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.
Topics: Animals; Mice; Dynamins; Histidine; Hypoxia; Mitophagy; Ubiquitination
PubMed: 38480688
DOI: 10.1038/s41467-024-46385-7