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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 -
BioRxiv : the Preprint Server For... Dec 2023Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier protein SLC25A46 interacts with both the outer and...
Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier protein SLC25A46 interacts with both the outer and inner-membrane dynamin family GTPases Mfn1/2 and Opa1. While SLC25A46 levels are known affect mitochondrial morphology, how SLC25A46 interacts with Mfn1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass-spectrometry and AlphaFold 2 modeling to identify interfaces mediating a SLC25A46-Opa1-Mfn1/2 complex. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and the helical repeat 1 region of Mfn2 interacts with the SLC25A46 N-terminus. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance.
PubMed: 38234813
DOI: 10.1101/2023.12.29.573615 -
Cell Death & Disease Jan 2024Mitochondria are highly dynamic organelles capable of altering their sizes and shapes to maintain metabolic balance through coordinated fission and fusion processes. In...
Mitochondria are highly dynamic organelles capable of altering their sizes and shapes to maintain metabolic balance through coordinated fission and fusion processes. In various cancer types, mitochondrial hyperfragmentation has been frequently observed, contributing to the progression of cancer toward metastasis. Inverted formin 2 (INF2), which resides in the endoplasmic reticulum (ER), has been found to accelerate actin polymerization and drive mitochondrial fission. In this study, we demonstrate that INF2 expression is significantly upregulated in endometrial cancer (EC) and is associated with a poor prognosis in EC patients. INF2 promotes anchorage-dependent and independent EC cell growth in part by facilitating mitochondrial fission. Furthermore, in conditions of energy stress, AMP-activated protein kinase (AMPK) phosphorylates INF2 at Ser1077, leading to increased localization of INF2 to the ER and enhanced recruitment of the dynamin-related protein 1 (DRP1) to mitochondria. This AMPK-mediated phosphorylation of INF2 at Ser1077 facilitates mitochondrial division and promotes EC cell growth. Pathological examination using immunohistochemical analyses revealed a positive correlation between AMPK activity and phosphorylated INF2 (Ser1077) in EC specimens. Collectively, our findings uncover novel molecular mechanisms involving the AMPK-INF2 axis, which regulates mitochondrial dynamics and malignant cell growth in EC.
Topics: Female; Humans; AMP-Activated Protein Kinases; Dynamins; Endometrial Neoplasms; Formins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Phosphorylation
PubMed: 38233384
DOI: 10.1038/s41419-024-06431-0 -
International Journal of Molecular... Dec 2023Novel components of the mitochondrial fission machinery, mitochondrial dynamics proteins of 49 kDa (MiD49) and 51 kDa (MiD51), have been recently described, and their...
Novel components of the mitochondrial fission machinery, mitochondrial dynamics proteins of 49 kDa (MiD49) and 51 kDa (MiD51), have been recently described, and their potential therapeutic targets for treating cardiovascular disease have been shown, including acute myocardial infarction (AMI), anthracycline cardiomyopathy and pulmonary arterial hypertension (PAH). Here, we examined the role of MiD49 and MiD51 in atherosclerosis. MiD49/51 expression was increased in the aortic valve endothelial cells (ECs) of high-fat diet-induced atherosclerosis in ApoEmice and IL-8-induced human umbilical vein endothelial cells (HUVECs), which accelerated dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Silencing MiD49/51 reduced atherosclerotic plaque size, increased collagen content, and decreased the IL-8-induced adhesion and proliferation of HUVECs. MiD51 upregulation resulted from decreased microRNA (miR)-107 expression and increased hypoxia-inducible factor-1a (HIF-1a) expression. Treatment with miR-107 mimics decreased atherosclerotic plaque size by reducing HIF-1α and MiD51 production. Both MiD49 and MiD51 were involved in atherosclerotic plaque formation through Drp1-mediated mitochondrial fission, and the involvement of MiD51 in this process was the result of decreased miR-107 expression and increased HIF-1α expression. The miR-107-HIF-1α-MiD51 pathway might provide new therapeutic targets for atherosclerosis.
Topics: Humans; Animals; Mice; Plaque, Atherosclerotic; Mitochondrial Dynamics; Diet, High-Fat; Interleukin-8; Atherosclerosis; Apolipoproteins E; Myocardial Infarction; Dynamins; Human Umbilical Vein Endothelial Cells; Mitochondrial Proteins; MicroRNAs
PubMed: 38203413
DOI: 10.3390/ijms25010244 -
Nanomedicine : Nanotechnology, Biology,... Feb 2024Anti-cancer monoclonal antibodies often fail to provide therapeutic benefit in receptor-positive patients due to rapid endocytosis of antibody-bound cell surface...
A PEGylated liposomal formulation of prochlorperazine that limits brain exposure but retains dynamin II activity: A potential adjuvant therapy for cancer patients receiving chemotherapeutic mAbs.
Anti-cancer monoclonal antibodies often fail to provide therapeutic benefit in receptor-positive patients due to rapid endocytosis of antibody-bound cell surface receptors. High dose co-administration of prochlorperazine (PCZ) inhibits endocytosis and sensitises tumours to mAbs by inhibiting dynamin II but can also introduce neurological side effects. We examined the potential to use PEGylated liposomal formulations of PCZ (LPCZ) to retain the anti-cancer effects of PCZ, but limit brain uptake. Uncharged liposomes showed complete drug encapsulation and pH-dependent drug release, but cationic liposomes showed limited drug encapsulation and lacked pH-dependent drug release. Uncharged LPCZ showed comparable inhibition of EGFR internalisation to free PCZ in KJD cells. After IV administration to rats, LPCZ reduced the plasma clearance and brain uptake of PCZ compared to IV PCZ. The results suggest that LPCZ may offer some benefit over PCZ as an adjunct therapy in cancer patients receiving mAb treatment.
Topics: Humans; Rats; Animals; Prochlorperazine; Dynamin II; Liposomes; Neoplasms; Antineoplastic Agents; Antibodies, Monoclonal; Brain; Polyethylene Glycols
PubMed: 38199450
DOI: 10.1016/j.nano.2024.102733 -
BioRxiv : the Preprint Server For... Dec 2023Dynamin-related protein 1 (Drp1), a large GTPase, mediates mitochondrial fission. Increased Drp1-mediated fission permits accelerated mitosis, contributing to...
RATIONALE
Dynamin-related protein 1 (Drp1), a large GTPase, mediates mitochondrial fission. Increased Drp1-mediated fission permits accelerated mitosis, contributing to hyperproliferation of pulmonary artery smooth muscle cells (PASMC), which characterizes pulmonary arterial hypertension (PAH). We developed a Drp1 inhibitor, Drpitor1a, and tested its ability to regress PAH.
OBJECTIVES
Assess Drpitor1a's efficacy and toxicity in: a)normal and PAH human PASMC (hPASMC); b)normal rats versus rats with established monocrotaline (MCT)-induced PAH.
METHODS
Drpitor1a's effects on recombinant and endogenous Drp1-GTPase activity, mitochondrial fission, and cell proliferation were studied in hPASMCs (normal=3; PAH=5). Drpitor1a's pharmacokinetics and tissue concentrations were measured (n=3 rats/sex). In a pilot study (n=3-4/sex/dose), Drpitor1a (1mg/kg/48-hours, intravenous) reduced adverse PA remodeling only in females. Consequently, we compared Drpitor1a to vehicle in normal (n=6 versus 8) and MCT-PAH (n=9 and 11) females, respectively. Drpitor1a treatment began 17-days post-MCT with echocardiography and cardiac catheterization performed 28-29 days post-MCT.
RESULTS
Drpitor1a inhibited recombinant and endogenous Drp1 GTPase activity, which was increased in PAH hPASMC. Drpitor1a inhibited mitochondrial fission and proliferation and induced apoptosis, in PAH hPASMC but not normal hPASMC. Drpitor1a tissue levels were higher in female versus male RVs. In MCT-PAH females, Drpitor1a regressed PA obstruction, lowered pulmonary vascular resistance, and improved RV function, without hematologic, renal, or hepatic toxicity.
CONCLUSIONS
Drpitor1a inhibits Drp1 GTPase, reduces mitochondrial fission, and inhibits cell proliferation in PAH hPASMC. Drpitor1a caused no toxicity in MCT-PAH and had no significant effect on normal rats or hPASMCs. Drpitor1a is a potential PAH therapeutic which displays an interesting therapeutic sexual dimorphism.
PubMed: 38187628
DOI: 10.1101/2023.12.21.572836 -
Nature Communications Jan 2024The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find...
The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.
Topics: Dynamins; GTP Phosphohydrolases; Mitochondria; Hydrolysis; Membrane Fusion; Mitochondrial Dynamics; Mitochondrial Proteins
PubMed: 38168038
DOI: 10.1038/s41467-023-44413-6 -
Nature Communications Jan 2024Membrane fusion and budding mediate fundamental processes like intracellular trafficking, exocytosis, and endocytosis. Fusion is thought to open a nanometer-range pore... (Review)
Review
Membrane fusion and budding mediate fundamental processes like intracellular trafficking, exocytosis, and endocytosis. Fusion is thought to open a nanometer-range pore that may subsequently close or dilate irreversibly, whereas budding transforms flat membranes into vesicles. Reviewing recent breakthroughs in real-time visualization of membrane transformations well exceeding this classical view, we synthesize a new model and describe its underlying mechanistic principles and functions. Fusion involves hemi-to-full fusion, pore expansion, constriction and/or closure while fusing vesicles may shrink, enlarge, or receive another vesicle fusion; endocytosis follows exocytosis primarily by closing Ω-shaped profiles pre-formed through the flat-to-Λ-to-Ω-shape transition or formed via fusion. Calcium/SNARE-dependent fusion machinery, cytoskeleton-dependent membrane tension, osmotic pressure, calcium/dynamin-dependent fission machinery, and actin/dynamin-dependent force machinery work together to generate fusion and budding modes differing in pore status, vesicle size, speed and quantity, controls release probability, synchronization and content release rates/amounts, and underlies exo-endocytosis coupling to maintain membrane homeostasis. These transformations, underlying mechanisms, and functions may be conserved for fusion and budding in general.
Topics: Cell Membrane; Calcium; Membrane Fusion; Exocytosis; Dynamins; Secretory Vesicles
PubMed: 38167896
DOI: 10.1038/s41467-023-44539-7 -
Analytical Cellular Pathology... 2023Nonobstructive azoospermia (NOA) is a complex disease characterized by the spermatogenic dysfunction of testicular tissues. The roles played by long noncoding RNAs...
BACKGROUND
Nonobstructive azoospermia (NOA) is a complex disease characterized by the spermatogenic dysfunction of testicular tissues. The roles played by long noncoding RNAs (lncRNAs) in NOA pathogenesis have not been extensively studied.
METHODS
Microarray assays were performed on samples of testicular biopsy tissue obtained from patients with NOA for the purpose of identifying differentially expressed lncRNAs and messenger RNA (mRNA) transcripts, and the results were verified by quantitative real-time polymerase chain reaction. Mouse-derived GC-1 spermatogonia (spg) cells undergoing treatment with Adriamycin (ADR) were used to investigate the biological functions of the selected lncRNAs . The target microRNAs (miRNAs) of lncRNAs and the target mRNAs of miRNAs were predicted by a bioinformatics analysis. Functional studies performed using the CCK-8 assay, EdU incorporation assay, apoptosis detection, and senescence-associated -galactosidase (SA--Gal) staining were conducted using GC-1 spg cells.
RESULTS
Totals of 2,652 lncRNAs and 2,625 mRNAs were found to be differentially expressed in the testicular tissue of NOA patients when compared with patients in a control group. Dynamin 3 opposite strand (DNM3OS) was a provider of pe-miR-214-5p that positively regulates miR-214-5p expression in GC-1 spg cells. The E2 factor (E2F) family of transcription factor 2 (E2F2) was initially predicted and subsequently verified to be a downstream gene of miR-214-5p. E2F2 expression was upregulated after DNM3OS knockdown in ADR-treated GC-1 spg cells. Moreover, knockdown of either DNM3OS or miR-214-5p significantly alleviated ADR-induced decreases in cellular activity and proliferation, as well as increases in apoptosis and senescence of mouse spermatogonial GC-1 spg cells.
CONCLUSIONS
DNM3OS was found to regulate the apoptosis and senescence of spermatogonia by providing miR-214-5p and decreasing E2F2 expression, suggesting it as a novel target for gene therapy of male infertility.
Topics: Animals; Humans; Male; Mice; Apoptosis; Azoospermia; Cell Proliferation; Dynamin III; E2F2 Transcription Factor; MicroRNAs; RNA, Long Noncoding; Spermatogonia; RNA, Antisense
PubMed: 38152068
DOI: 10.1155/2023/1477658 -
Annals of Clinical and Translational... Mar 2024Movement disorders manifest in various hereditary neurodegenerative diseases. We reported a young man who presented with progressive upper limb dystonia, spastic...
Movement disorders manifest in various hereditary neurodegenerative diseases. We reported a young man who presented with progressive upper limb dystonia, spastic tetraplegia, and ataxia. Whole-exome sequencing (WES) revealed a novel variant, c.2357A > G, in the dynamin domain of OPA1. No mtDNA deletion was detected in muscle by long-range PCR. Atrophy and decreased glucose metabolism of the basal ganglia were discovered. Decreased mtDNA copy number, fragmented mitochondria, slightly impaired oxidative phosphorylation, and increased autophagy were detected in mutant fibroblasts. Evident oxidative phosphorylation impairment and mtDNA deletions were not involved in the pathogenicity of this mutation unlike mutations in the GTPase domain of OPA1.
Topics: Male; Humans; Dystonia; Dynamins; Mutation; Ataxia; DNA, Mitochondrial; Quadriplegia; GTP Phosphohydrolases
PubMed: 38148580
DOI: 10.1002/acn3.51981