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Progress in Molecular Biology and... 2013Proteins of the dynamin superfamily are mechanochemical GTPases, which mediate nucleotide-dependent membrane remodeling events. The founding member dynamin is recruited... (Review)
Review
Proteins of the dynamin superfamily are mechanochemical GTPases, which mediate nucleotide-dependent membrane remodeling events. The founding member dynamin is recruited to the neck of clathrin-coated endocytic vesicles where it oligomerizes into helical filaments. Nucleotide-hydrolysis-induced conformational changes in the oligomer catalyze scission of the vesicle neck. Here, we review recent insights into structure, function, and oligomerization of dynamin superfamily proteins and their roles in human diseases. We describe in detail the molecular mechanisms how dynamin oligomerizes at membranes and introduce a model how oligomerization is linked to membrane fission. Finally, we discuss molecular mechanisms how mutations in dynamin could lead to the congenital diseases, Centronuclear Myopathy and Charcot-Marie Tooth disease.
Topics: Animals; Disease; Dynamins; Health; Humans; Models, Molecular; Mutation; Protein Multimerization
PubMed: 23663977
DOI: 10.1016/B978-0-12-386931-9.00015-5 -
Neurotherapeutics : the Journal of the... Oct 2018Dynamin 2 (DNM2) belongs to a family of large GTPases that are well known for mediating membrane fission by oligomerizing at the neck of membrane invaginations.... (Review)
Review
Dynamin 2 (DNM2) belongs to a family of large GTPases that are well known for mediating membrane fission by oligomerizing at the neck of membrane invaginations. Autosomal dominant mutations in the ubiquitously expressed DNM2 cause 2 discrete neuromuscular diseases: autosomal dominant centronuclear myopathy (ADCNM) and dominant intermediate Charcot-Marie-Tooth neuropathy (CMT). CNM and CMT mutations may affect DNM2 in distinct manners: CNM mutations may cause protein hyperactivity with elevated GTPase and fission activities, while CMT mutations could impair DNM2 lipid binding and activity. DNM2 is also a modifier of the X-linked and autosomal recessive forms of CNM, as DNM2 protein levels are upregulated in animal models and patient muscle samples. Strikingly, reducing DNM2 has been shown to revert muscle phenotypes in preclinical models of CNM. As DNM2 emerges as the key player in CNM pathogenesis, the role(s) of DNM2 in skeletal muscle remains unclear. This review aims to provide insights into potential pathomechanisms related to DNM2-CNM mutations, and discuss exciting outcomes of current and future therapeutic approaches targeting DNM2 hyperactivity.
Topics: Animals; Dynamin II; Dynamins; Humans; Muscle, Skeletal; Mutation; Neuromuscular Diseases
PubMed: 30426359
DOI: 10.1007/s13311-018-00686-0 -
Biophysical Journal Feb 2011Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ∼40 kDa SH3 domain-containing protein that binds to the...
Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ∼40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin, a ∼100 kDa GTPase that is essential for endocytic membrane scission. It has been suggested that endophilin is monomeric in the cytoplasm and dimerizes only after it binds to membranes (or perhaps to dimers or tetramers of dynamin). To clarify this issue, we studied the oligomeric state of endophilin both in vitro using analytical ultracentrifugation and fluorescence anisotropy, and in living cells using two-photon fluorescence fluctuation spectroscopy. We analyzed the fluctuation data using the Q-analysis method, which allowed us to determine the intrinsic brightness of the labeled protein complexes and hence its aggregation state in the cytoplasmic regions of the cell. Although a relatively high K(d) (∼5-15 μM) was observed in vitro, the cell measurements indicate that endophilin is dimeric in the cytoplasm, even at submicromolar concentrations. We also demonstrate that endophilin significantly enhances the assembly of dynamin, and that this enhancement is proportional to the fraction of dimeric endophilin that is present. Moreover, there is correlation between the concentrations of endophilin that promote dynamin self-assembly and those that stimulate dynamin GTPase activity. These findings support the view that endophilin-dynamin interactions play an important role in endocytosis.
Topics: Animals; Anisotropy; Cell Survival; Dynamin II; Green Fluorescent Proteins; Intracellular Signaling Peptides and Proteins; Models, Molecular; Nephelometry and Turbidimetry; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Rats; Scattering, Small Angle; Solutions; Spectrometry, Fluorescence; X-Ray Diffraction; src Homology Domains
PubMed: 21281588
DOI: 10.1016/j.bpj.2010.12.3717 -
ChemMedChem Jul 2009Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospholipid-stimulated GTPase activity of dynamin I. Analogues of Bis-T have...
Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospholipid-stimulated GTPase activity of dynamin I. Analogues of Bis-T have significant potential as a biological probes for the dissection of endocytic pathways. Bis-T-derived compounds were synthesised and evaluated for their ability to inhibit the GTPase activity of dynamin I. Two analogues (23 and 24) represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibition.Two azidobenzyl amide (4 and 23) and one 3-trifluoromethyl-3H-diazirin-3-ylphenyl (24) analogues of bis-tyrphostin (1, Bis-T) were synthesised as potential photoaffinity labels for the elucidation of the binding site of compound 1 in dynamin I. Of the two azidobenzyl amide analogues (4 and 23), the terminally substituted 23 retained dynamin I GTPase inhibition (IC(50)=6.4+/-2.8 microM) whilst 4, which was substituted on the central carbon of the amide linker, displayed no activity. Analogue 24 also retained inhibitory activity (IC(50)=36+/-9 microM). Photoaffinity labelling experiments did not unequivocally elucidate the binding pocket of compound 1. However, compounds 23 and 24 represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibitory activity, providing a new direction for analogue synthesis.
Topics: Animals; Dynamin I; Enzyme Inhibitors; Sheep; Tyrphostins
PubMed: 19437476
DOI: 10.1002/cmdc.200900054 -
Trends in Cell Biology Mar 1999The function of the GTPase dynamin has been discussed for several years. It clearly plays a role in vesicle budding, but, despite recent insights, precisely how it... (Review)
Review
The function of the GTPase dynamin has been discussed for several years. It clearly plays a role in vesicle budding, but, despite recent insights, precisely how it functions in this process is still a matter of debate. In addition, it is now clear that dynamin is a member of a large protein family, present in a variety of cellular locations where members apparently perform a range of functions. This article describes current understanding of the structure and function of the various dynamin family members.
Topics: Amino Acid Sequence; Animals; Dynamins; GTP Phosphohydrolases; Humans; Molecular Sequence Data
PubMed: 10201074
DOI: 10.1016/s0962-8924(98)01490-1 -
Nature Dec 2006Dynamins form a superfamily of large mechano-chemical GTPases that includes the classical dynamins and dynamin-like proteins (DLPs). They are found throughout the...
Dynamins form a superfamily of large mechano-chemical GTPases that includes the classical dynamins and dynamin-like proteins (DLPs). They are found throughout the Eukarya, functioning in core cellular processes such as endocytosis and organelle division. Many bacteria are predicted by sequence to possess large GTPases with the same multidomain architecture that is found in DLPs. Mechanistic dissection of dynamin family members has been impeded by a lack of high-resolution structural data currently restricted to the GTPase and pleckstrin homology domains, and the dynamin-related human guanylate-binding protein. Here we present the crystal structure of a cyanobacterial DLP in both nucleotide-free and GDP-associated conformation. The bacterial DLP shows dynamin-like qualities, such as helical self-assembly and tubulation of a lipid bilayer. In vivo, it localizes to the membrane in a manner reminiscent of FZL, a chloroplast-specific dynamin-related protein with which it shares sequence similarity. Our results provide structural and mechanistic insight that may be relevant across the dynamin superfamily. Concurrently, we show compelling similarity between a cyanobacterial and chloroplast DLP that, given the endosymbiotic ancestry of chloroplasts, questions the evolutionary origins of dynamins.
Topics: Animals; Bacterial Proteins; Crystallography, X-Ray; Dynamins; Liposomes; Nostoc
PubMed: 17122778
DOI: 10.1038/nature05312 -
European Journal of Medicinal Chemistry Oct 2014Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4-benzoquinone 1 as...
Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4-benzoquinone 1 as a 273 ± 106 μM inhibitor. In silico lead optimization and focused library-led synthesis resulted in the development of four discrete benzoquinone/naphthoquinone based compound libraries comprising 54 compounds in total. Sixteen analogues were more potent than lead 1, with 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone (45) and 2,5-bis(4-carboxyanilino)-1,4-benzoquinone (49) the most active with IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 μM respectively. Molecular modelling suggested a number of hydrogen bonding and hydrophobic interactions were involved in stabilization of 49 within the dynI GTP binding site. Six of the most active inhibitors were evaluated for potential inhibition of clathrin-mediated endocytosis (CME). Quinone 45 was the most effective CME inhibitor with an IC50(CME) of 36 ± 16 μM.
Topics: Animals; Cell Line; Clathrin; Drug Evaluation, Preclinical; Dynamin I; Endocytosis; Enzyme Inhibitors; GTP Phosphohydrolases; Humans; Molecular Docking Simulation; Protein Conformation; Quinones; Structure-Activity Relationship
PubMed: 25084145
DOI: 10.1016/j.ejmech.2014.06.070 -
Cell Reports Aug 2014Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the...
Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, which is necessary for efficient membrane fission during endocytosis. Recent evidence suggests that the transition state of dynamin's GTP hydrolysis reaction serves as a key determinant of productive fission. Here, we present the structure of a transition-state-defective dynamin mutant K44A trapped in a prefission state at 12.5 Å resolution. This structure constricts to 3.7 nm, reaching the theoretical limit required for spontaneous membrane fission. Computational docking indicates that the ground-state conformation of the dynamin polymer is sufficient to achieve this superconstricted prefission state and reveals how a two-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. These data suggest a model for the assembly and regulation of the minimal dynamin fission machine.
Topics: Amino Acid Sequence; Cell Membrane; Clathrin-Coated Vesicles; Dynamins; Guanosine Triphosphate; Humans; Molecular Dynamics Simulation; Molecular Sequence Data; Mutation; Protein Multimerization; Protein Structure, Tertiary
PubMed: 25088425
DOI: 10.1016/j.celrep.2014.06.054 -
Methods in Enzymology 2005Dynamin, unlike many GTPase superfamily members, exhibits a relatively rapid basal rate of GTP hydrolysis that is not rate-limited by GTP binding or GDP dissociation....
Dynamin, unlike many GTPase superfamily members, exhibits a relatively rapid basal rate of GTP hydrolysis that is not rate-limited by GTP binding or GDP dissociation. Also unique to dynamin GTPase family members is their ability to self-assemble into rings and helical stacks of rings either in solution or onto lipid templates. Self-assembly stimulates dynamin's GTPase activity by >100-fold. Given these robust rates of GTP hydrolysis compared to most GTPases, GTP hydrolysis by dynamin can be easily measured using a simple colorimetic assay to detect released phosphate. We describe this assay and report variations in assay conditions that have contributed to the wide range of reported values for dynamin's basal and assembly-stimulated rates of GTP hydrolysis.
Topics: Animals; Colorimetry; Dynamins; GTP Phosphohydrolases; Liposomes; Nanotubes; Recombinant Proteins; Spodoptera
PubMed: 16413294
DOI: 10.1016/S0076-6879(05)04043-7 -
The Journal of Biological Chemistry Jul 1998To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin-1 wild type and a GTPase-negative mutant (K44A) in primary rat...
To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin-1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. Transfection was accomplished by electroporation using an hemagglutinin (HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4 alone, insulin results in an approximately 7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of HA-GLUT4-trafficking parallel those of the native GLUT4 and in addition, that newly synthesized HA-GLUT4 goes to the plasma membrane before being sorted into the insulin-responsive compartments. Short term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. Under conditions of maximal expression of dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose transporters. In contrast, expression of dynamin wild type decreases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. These data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin.
Topics: Adipocytes; Animals; Dynamin I; Dynamins; Electroporation; Endocytosis; GTP Phosphohydrolases; Glucose Transporter Type 4; Hemagglutinins; Insulin; Kinetics; Male; Monosaccharide Transport Proteins; Muscle Proteins; Rats
PubMed: 9651341
DOI: 10.1074/jbc.273.28.17504