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Redox Biology Jul 2023Silver nanoparticles (AgNPs) have widely used in industrial and medical applications for their excellent antibacterial activities. AgNPs can penetrate into the brain and...
Silver nanoparticles (AgNPs) have widely used in industrial and medical applications for their excellent antibacterial activities. AgNPs can penetrate into the brain and cause neuronal death, but limited evidence focused on toxic effects and mechanic study in hippocampal neuron. This study aimed to investigate the molecular mechanisms of mitochondrial damage and apoptosis in mouse hippocampal HT22 cells and further to explore role of reactive oxygen species (ROS) and GTPase dynamin-related protein 1 (Drp1) in AgNPs-induced neurotoxicity. Our results showed that acute exposure to AgNPs at low doses (2-8 μg/mL) increased ROS generation, decreased mitochondrial membrane potential (MMP) and ATP synthesis in HT22 cells. In addition, AgNPs promoted mitochondrial fragmentation and mitochondria-dependent apoptosis via excessive mitochondrial fission/fusion by 8 μg/mL AgNPs treatment for 24 h. The mechanism was involved in increased protein expression of Drp1, mitochondrial fission protein 1 (Fis1), mitofusin 1/2 (Mfn1/2) and inhibited optic atrophy 1 (OPA1), and mainly mediated by phosphorylation of Drp1 Ser616. The AgNPs-induced mitochondrial impairment and apoptosis was mainly due to their particle-specific effect rather than silver ions release. Furthermore Drp1-mediated mitochondrial fission contributed to mitochondria-dependent apoptosis induced by AgNPs, all aforementioned changes were significantly rescued by N-acetyl-l-cysteine (NAC) and Mdivi-1 except for OPA1 protein expression. Hence, our results provide a novel neurotoxic mechanism to AgNPs-induced neurotoxicity and revealed that the mechanism of mitochondria-dependent apoptosis in HT22 cells was mediated by excessive activation of ROS-Drp1-mitochondrial fission axis. These findings can deepen current evidences on neurotoxicological evaluation of AgNPs and aid in guiding their proper applications in different areas, especially in biomedical use.
Topics: Mice; Animals; Reactive Oxygen Species; Silver; Metal Nanoparticles; Dynamins; Apoptosis; Mitochondria; Hippocampus; Mitochondrial Dynamics
PubMed: 37187014
DOI: 10.1016/j.redox.2023.102739 -
PloS One 2017Clathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key...
Clathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamin's GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamin's stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamin's basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several "hits" that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin's basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for more potent and chemically desirable dynamin inhibitors.
Topics: Dynamins; Fluorescence Polarization; GTP Phosphohydrolases; High-Throughput Screening Assays; Limit of Detection
PubMed: 28957392
DOI: 10.1371/journal.pone.0185639 -
ChemMedChem Jan 2022Five focused libraries of pyrimidine-based dynamin GTPase inhibitors, in total 69 compounds were synthesised, and their dynamin inhibition and broad-spectrum...
Five focused libraries of pyrimidine-based dynamin GTPase inhibitors, in total 69 compounds were synthesised, and their dynamin inhibition and broad-spectrum cytotoxicity examined. Dynamin plays a crucial role in mitosis, and as such inhibition of dynamin was expected to broadly correlate with the observed cytotoxicity. The pyrimidines synthesised ranged from mono-substituted to trisubstituted. The highest levels of dynamin inhibition were noted with di- and tri- substituted pyrimidines, especially those with pendent amino alkyl chains. Short chains and simple heterocyclic rings reduced dynamin activity. There were three levels of dynamin activity noted: 1-10, 10-25 and 25-60 μM. Screening of these compounds in a panel of cancer cell lines: SW480 (colon), HT29 (colon), SMA (spontaneous murine astrocytoma), MCF-7 (breast), BE2-C (glioblastoma), SJ-G2 (neuroblastoma), MIA (pancreas), A2780 (ovarian), A431 (skin), H460 (lung), U87 (glioblastoma) and DU145 (prostate) cell lines reveal a good correlation between the observed dynamin inhibition and the observed cytotoxicity. The most active analogues (31 a,b) developed returned average GI values of 1.0 and 0.78 μM across the twelve cell lines examined. These active analogues were: N -(3-dimethylaminopropyl)-N -dodecyl-6-methylpyrimidine-2,4-diamine (31 a) and N -(3-dimethylaminopropyl)-N -dodecyl-6-methylpyrimidine-2,4-diamine (31 b).
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cytotoxins; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Dynamins; Enzyme Inhibitors; Humans; Molecular Structure; Pyrimidines; Structure-Activity Relationship
PubMed: 34590434
DOI: 10.1002/cmdc.202100560 -
International Review of Cell and... 2013The large GTPase dynamin is well known for its actions on budded cellular membranes to generate vesicles, most often, clathrin-coated endocytic vesicles. The scope of... (Review)
Review
The large GTPase dynamin is well known for its actions on budded cellular membranes to generate vesicles, most often, clathrin-coated endocytic vesicles. The scope of cellular processes in which dynamin-mediated vesicle formation occurs, has expanded to include secretory vesicle formation at the Golgi, from other endosomes and nonclathrin structures, such as caveolae, as well as membrane remodeling during exocytosis and vesicle fusion. An intriguing new facet of dynamin's sphere of influence is the cytoskeleton. Cytoskeletal filament networks maintain cell shape, provide cell movement, execute cell division and orchestrate vesicle trafficking. Recent evidence supports the hypothesis that dynamin influences actin filaments and microtubules via mechanisms that are independent of its membrane-remodeling activities. This chapter discusses this emerging evidence and considers possible mechanisms of action.
Topics: Actin Cytoskeleton; Biological Transport, Active; Caveolae; Cell Division; Dynamins; Eukaryotic Cells; Exocytosis; Membrane Fusion
PubMed: 23351711
DOI: 10.1016/B978-0-12-407699-0.00003-0 -
The EMBO Journal Sep 2010The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on...
The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on phosphoinositide-containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin-homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C-terminal α-helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either 'sensitize' dynamin to lipid stimulation or elevate basal GTPase rates by promoting self-assembly and thus rendering dynamin no longer lipid responsive. We also describe a low-resolution structure of dimeric dynamin from small-angle X-ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self-assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.
Topics: Blood Proteins; Dynamins; GTP Phosphohydrolases; Humans; Hydrolysis; Membrane Lipids; Mutation; Myopathies, Structural, Congenital; Phosphoproteins; Protein Structure, Tertiary; X-Ray Diffraction
PubMed: 20700106
DOI: 10.1038/emboj.2010.187 -
Molecular Biology of the Cell Sep 2001Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation...
Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.
Topics: Amino Acid Substitution; Animals; Cell Line, Transformed; Coated Pits, Cell-Membrane; Dynamins; Endocytosis; GTP Phosphohydrolases; Genes, Dominant; Guanosine Triphosphate; HeLa Cells; Humans; Kinetics; Mice; Microscopy, Electron; Point Mutation; Protein Structure, Tertiary; Transport Vesicles
PubMed: 11553700
DOI: 10.1091/mbc.12.9.2578 -
Journal of Interferon & Cytokine... Aug 2005The guanylate-binding proteins (GBPs) were first identified in the late 1970s, and within a short period of time, investigators were aware that GBPs possessed unique... (Review)
Review
The guanylate-binding proteins (GBPs) were first identified in the late 1970s, and within a short period of time, investigators were aware that GBPs possessed unique properties, in particular the ability to bind GMP agarose. Since then, much study has gone into understanding their mechanism of induction by interferons (IFNs) and other cytokines, and they have been used extensively as markers for IFN responsiveness in both cells and organisms. In time, we learned that GBPs had the unusual ability to hydrolyze GTP to both GDP and GMP. More recently, we have begun to appreciate their novel structure, one that suggests unique mechanisms of GTP binding and hydrolysis and unique forms of regulation. In addition, we have begun to unravel some of their functions and to separate these function into those functions that do and those that do not require GTPase activity.
Topics: Animals; Cytokines; Dynamins; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Inflammation; Protein Prenylation
PubMed: 16108726
DOI: 10.1089/jir.2005.25.435 -
The International Journal of... Jan 2014Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the...
Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0-21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation.
Topics: Animals; Cell Differentiation; Cell Movement; Dynamins; Gene Knockdown Techniques; HEK293 Cells; Humans; Mice; Osteoblasts; Phosphorylation
PubMed: 24387844
DOI: 10.1016/j.biocel.2013.10.008 -
Traffic (Copenhagen, Denmark) Mar 2006Animal and plant cytokineses appear morphologically distinct. Recent studies, however, have revealed that these cellular processes have many things in common, including... (Review)
Review
Animal and plant cytokineses appear morphologically distinct. Recent studies, however, have revealed that these cellular processes have many things in common, including the requirement of co-ordinated membrane trafficking and cytoskeletal dynamics. At the intersection of these two processes are the members of the dynamin family of ubiquitous eukaryotic GTPases. In this review, we highlight the conserved contribution of classical dynamin and dynamin-related proteins during cytokinesis in both animal and plant systems.
Topics: Animals; Biological Transport; Cell Division; Cell Membrane; Cytokinesis; Dynamins; Endocytosis; Models, Biological; Plant Proteins; Plants; Protein Structure, Tertiary; Protein Transport
PubMed: 16497219
DOI: 10.1111/j.1600-0854.2006.00385.x -
FASEB Journal : Official Publication of... Jan 2020Mitochondrial fission is important in physiological processes, including coordination of mitochondrial and nuclear division during mitosis, and pathologic processes,...
Identification of novel dynamin-related protein 1 (Drp1) GTPase inhibitors: Therapeutic potential of Drpitor1 and Drpitor1a in cancer and cardiac ischemia-reperfusion injury.
Mitochondrial fission is important in physiological processes, including coordination of mitochondrial and nuclear division during mitosis, and pathologic processes, such as the production of reactive oxygen species (ROS) during cardiac ischemia-reperfusion injury (IR). Mitochondrial fission is mainly mediated by dynamin-related protein 1 (Drp1), a large GTPase. The GTPase activity of Drp1 is essential for its fissogenic activity. Therefore, we aimed to identify Drp1 inhibitors and evaluate their anti-neoplastic and cardioprotective properties in five cancer cell lines (A549, SK-MES-1, SK-LU-1, SW 900, and MCF7) and an experimental cardiac IR injury model. Virtual screening of a chemical library revealed 17 compounds with high predicted affinity to the GTPase domain of Drp1. In silico screening identified an ellipticine compound, Drpitor1, as a putative, potent Drp1 inhibitor. We also synthesized a congener of Drpitor1 to remove the methoxymethyl group and reduce hydrolytic lability (Drpitor1a). Drpitor1 and Drpitor1a inhibited the GTPase activity of Drp1 without inhibiting the GTPase of dynamin 1. Drpitor1 and Drpitor1a have greater potency than the current standard Drp1 GTPase inhibitor, mdivi-1, (IC50 for mitochondrial fragmentation are 0.09, 0.06, and 10 μM, respectively). Both Drpitors reduced proliferation and induced apoptosis in cancer cells. Drpitor1a suppressed lung cancer tumor growth in a mouse xenograft model. Drpitor1a also inhibited mitochondrial ROS production, prevented mitochondrial fission, and improved right ventricular diastolic dysfunction during IR injury. In conclusion, Drpitors are useful tools for understanding mitochondrial dynamics and have therapeutic potential in treating cancer and cardiac IR injury.
Topics: A549 Cells; Animals; Dynamins; Enzyme Inhibitors; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Knockout; Myocardial Reperfusion Injury; Neoplasms; Rats; Rats, Sprague-Dawley; Xenograft Model Antitumor Assays
PubMed: 31914641
DOI: 10.1096/fj.201901467R