RESUMEN
BACKGROUND: Hereditary Spastic Paraplegia (HSP) represents a large group of clinically and genetically heterogeneous disorders linked to over 70 different loci and more than 60 recognized disease-causing genes. A heightened vulnerability to disruption of various cellular processes inherent to the unique function and morphology of corticospinal neurons may account, at least in part, for the genetic heterogeneity. METHODS: Whole exome sequencing was utilized to identify candidate genetic variants in a four-generation Siberian kindred that includes nine individuals showing clinical features of HSP. Segregation of candidate variants within the family yielded a disease-associated mutation. Functional as well as in-silico structural analyses confirmed the selected candidate variant to be causative. RESULTS: Nine known patients had young-adult onset of bilateral slowly progressive lower-limb spasticity, weakness and hyperreflexia progressing over two-to-three decades to wheel-chair dependency. In the advanced stage of the disease, some patients also had distal wasting of lower leg muscles, pes cavus, mildly decreased vibratory sense in the ankles, and urinary urgency along with electrophysiological evidence of a mild distal motor/sensory axonopathy. Molecular analyses uncovered a missense c.2155C > T, p.R719W mutation in the highly conserved GTP-effector domain of dynamin 2. The mutant DNM2 co-segregated with HSP and affected endocytosis when expressed in HeLa cells. In-silico modeling indicated that this HSP-associated dynamin 2 mutation is located in a highly conserved bundle-signaling element of the protein while dynamin 2 mutations associated with other disorders are located in the stalk and PH domains; p.R719W potentially disrupts dynamin 2 assembly. CONCLUSION: This is the first report linking a mutation in dynamin 2 to a HSP phenotype. Dynamin 2 mutations have previously been associated with other phenotypes including two forms of Charcot-Marie-Tooth neuropathy and centronuclear myopathy. These strikingly different pathogenic effects may depend on structural relationships the mutations disrupt. Awareness of this distinct association between HSP and c.2155C > T, p.R719W mutation will facilitate ascertainment of additional DNM2 HSP families and will direct future research toward better understanding of cell biological processes involved in these partly overlapping clinical syndromes.
Asunto(s)
Dinaminas/genética , Exoma , GTP Fosfohidrolasas/genética , Paraplejía Espástica Hereditaria/genética , Adulto , Análisis Mutacional de ADN , Dinamina II , Salud de la Familia , Femenino , GTP Fosfohidrolasas/química , Variación Genética , Células HeLa , Humanos , Masculino , Persona de Mediana Edad , Mutación , Mutación Missense , Fenotipo , SiberiaRESUMEN
Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate, characterized by a 'stalk' in which only the outer membrane monolayers of the two compartments have merged to form a localized non-bilayer connection. Formation of the hemi-fission intermediate requires energy input from proteins catalysing membrane remodelling; however, the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analysed how the GTPase cycle of human dynamin 1, the prototypical membrane fission catalyst, is directly coupled to membrane remodelling. We used intramolecular chemical crosslinking to stabilize dynamin in its GDP·AlF4(-)-bound transition state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fuelled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent, drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction, the force bimodality might constitute a general paradigm for leakage-free membrane remodelling.
Asunto(s)
Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Dinamina I/metabolismo , Biocatálisis , Proteínas Sanguíneas/química , Dinamina I/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Fusión de Membrana , Modelos Moleculares , Fosfoproteínas/química , Conformación ProteicaRESUMEN
Dynamins and BAR proteins are crucial in a wide variety of cellular processes for their ability to mediate membrane remodeling, such as membrane curvature and membrane fission and fusion. In this review, we highlight dynamins and BAR proteins and the cellular mechanisms that are involved in the initiation and progression of cancer. We specifically discuss the roles of the seproteinsin endocytosis, endo-lysosomal trafficking, autophagy, and apoptosis as these processes are all tightly linked to membrane remodeling and cancer.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Dinaminas/metabolismo , Neoplasias/metabolismo , Animales , Apoptosis , Autofagia , Endocitosis , Femenino , GTP Fosfohidrolasas/metabolismo , Humanos , Masculino , Neoplasias/fisiopatologíaRESUMEN
Dynamin is a large GTPase that mediates plasma membrane fission during clathrin-mediated endocytosis. Dynamin assembles into polymers on the necks of budding membranes in cells and has been shown to undergo GTP-dependent conformational changes that lead to membrane fission in vitro. Recent efforts have shed new light on the mechanisms of dynamin-mediated fission, yet exactly how dynamin performs this function in vivo is still not fully understood. Dynamin interacts with a number of proteins during the endocytic process. These interactions are mediated by the C-terminal proline-rich domain (PRD) of dynamin binding to SH3 domain-containing proteins. Three of these dynamin-binding partners (intersectin, amphiphysin and endophilin) have been shown to play important roles in the clathrin-mediated endocytosis process. They promote dynamin-mediated plasma membrane fission by regulating three important sequential steps in the process: recruitment of dynamin to sites of endocytosis; assembly of dynamin into a functional fission complex at the necks of clathrin-coated pits (CCPs); and regulation of dynamin-stimulated GTPase activity, a key requirement for fission.
RESUMEN
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.