Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 angstrom resolution. The hexagonal-shaped crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 angstrom, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

PLoS ONE / Exposure to indoor allergens in different residential settings and its influence on IgE sensitization in a geographically confined Austrian cohort

Background Exposure to indoor allergens is crucial for IgE sensitization and development of allergic symptoms. Residential settings influence the allergen amount in house dust and hence allergic sensitization. Within this study, we investigated allergen exposure and molecule-based IgE levels in a geographically confined region and evaluated the impact of housing, pets and cleaning. Methods 501 adolescents from Salzburg, Austria participated in this cross-sectional study. House dust samples were examined regarding major mite, cat, dog, and mold allergens using a multiplex assay. Serum samples of participants were analyzed for specific IgE to Der p 1, Der p 2, Fel d 1, Can f 1 and Alt a 1 using the multiplex array ImmunoCAP ISAC. Information on allergies, living areas, dwelling form (house, flat, farm), pets, and household cleanliness were obtained by a questionnaire. Results In investigated house dust samples, the concentration of cat allergen was highest while the prevalence of mold allergens was very low. Participants showed IgE sensitization to Der p 1 (13.2%), Der p 2 (18.2%), Fel d 1 (14.4%), Can f 1 (2.4%) and Alt a 1 (2.0%). In alpine regions, lower mite allergen concentrations were detected which correlated with reduced IgE levels. A trend for increased sensitization prevalence from rural to alpine to urban regions was noted. Living on farms resulted in lower sensitization prevalence to mite and cat allergens, even though exposure to mites was significantly elevated. The presence of cats was associated with a lower sensitization rate and IgE levels to cat and mite allergens, and less frequent allergic diseases. Cleaning did not impact allergen concentrations, while IgE reactivity to mites and allergic diseases were more pronounced when living in cleaner homes. Conclusion Allergen exposure to indoor allergens was influenced by setting of homes. Living in a farm environment and having a cat at home showed a pro tective effect for IgE sensitization and allergies. This cross-sectional study in combination with hereditary and lifestyle factors enables development of risk schemes for a more efficient management and potential prevention of allergic diseases

Mutant Huntingtin Binds The Mitochondrial Fission Gtpase Dynamin-Related Protein-1 And Increases Its Enzymatic Activity

Huntington\u27s disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington\u27s disease pathogenesis. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington\u27s disease in vitro and in a mouse model of Huntington\u27s disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington\u27s disease, which, in turn, stimulates its enzymatic activity. Mutant huntingtin-mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington\u27s disease. © 2011 Nature America, Inc. All rights reserved

S-Nitrosylation Of Drp1 Does Not Affect Enzymatic Activity And Is Not Specific To Alzheimer\u27S Disease

Mitochondrial dysfunction and synaptic loss are among the earliest events linked to Alzheimer\u27s disease (AD) and might play a causative role in disease onset and progression. The underlying mechanisms of mitochondrial and synaptic dysfunction in AD remain unclear. We previously reported that nitric oxide (NO) triggers persistent mitochondrial fission and causes neuronal cell death. A recent article claimed that S-nitrosylation of dynamin related protein 1 (DRP1) at cysteine 644 causes protein dimerization and increased GTPase activity and is the mechanism responsible for NO-induced mitochondrial fission and neuronal injury in AD, but not in Parkinson\u27s disease (PD). However, this report remains controversial. To resolve the controversy, we investigated the effects of S-nitrosylation on DRP1 structure and function. Contrary to the previous report, S-nitrosylation of DRP1 does not increase GTPase activity or cause dimerization. In fact, DRP1 does not exist as a dimer under native conditions, but rather as a tetramer capable of self-assembly into higher order spiral-and ring-like oligomeric structures after nucleotide binding. S-nitrosylation, as confirmed by the biotin-switch assay, has no impact on DRP1 oligomerization. Importantly, we found no significant difference in S-nitrosylated DRP1 (SNO-DRP1) levels in brains of age-matched normal, AD, or PD patients. We also found that S-nitrosylation is not specific to DRP1 because S-nitrosylated optic atrophy 1 (SNO-OPA1) is present at comparable levels in all human brain samples. Finally, we show that NO triggers DRP1 phosphorylation at serine 616, which results in its activation and recruitment to mitochondria. Our data indicate the mechanism underlying nitrosative stress-induced mitochondrial fragmentation in AD is not DRP1 S-nitrosylation. © 2010 IOS Press and the authors. All rights reserved

Functional mapping of human dynamin-1-like GTPase domain based on x-ray structure analyses.

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5'-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains

Exposure to Indoor Allergens in Different Residential Settings and Its Influence on IgE Sensitization in a Geographically Confined Austrian Cohort.

Exposure to indoor allergens is crucial for IgE sensitization and development of allergic symptoms. Residential settings influence the allergen amount in house dust and hence allergic sensitization. Within this study, we investigated allergen exposure and molecule-based IgE levels in a geographically confined region and evaluated the impact of housing, pets and cleaning.501 adolescents from Salzburg, Austria participated in this cross-sectional study. House dust samples were examined regarding major mite, cat, dog, and mold allergens using a multiplex assay. Serum samples of participants were analyzed for specific IgE to Der p 1, Der p 2, Fel d 1, Can f 1 and Alt a 1 using the multiplex array ImmunoCAP ISAC. Information on allergies, living areas, dwelling form (house, flat, farm), pets, and household cleanliness were obtained by a questionnaire.In investigated house dust samples, the concentration of cat allergen was highest while the prevalence of mold allergens was very low. Participants showed IgE sensitization to Der p 1 (13.2%), Der p 2 (18.2%), Fel d 1 (14.4%), Can f 1 (2.4%) and Alt a 1 (2.0%). In alpine regions, lower mite allergen concentrations were detected which correlated with reduced IgE levels. A trend for increased sensitization prevalence from rural to alpine to urban regions was noted. Living on farms resulted in lower sensitization prevalence to mite and cat allergens, even though exposure to mites was significantly elevated. The presence of cats was associated with a lower sensitization rate and IgE levels to cat and mite allergens, and less frequent allergic diseases. Cleaning did not impact allergen concentrations, while IgE reactivity to mites and allergic diseases were more pronounced when living in cleaner homes.Allergen exposure to indoor allergens was influenced by setting of homes. Living in a farm environment and having a cat at home showed a protective effect for IgE sensitization and allergies. This cross-sectional study in combination with hereditary and lifestyle factors enables development of risk schemes for a more efficient management and potential prevention of allergic diseases

Structure-function map of the modelled DNM1L active site dimer.

<p>All active site and dimerization residues that have been mutated to alanine are represented as stick models, as well as the GTP. The turnover numbers of the respective mutants as determined by the GTPase assay for basal activity are shown, whereby the WT was defined as 100%. Molecule A of the dimer is depicted in green, while the second molecule B is shown in orange, with the corresponding D190A*.</p

Kinetic parameters of DNM1L basal GTPase activities.

a<p>In all cases except for the cooperative model with the mutant S35A, k_obs and K correspond to k_cat and K_m of the applied Michaelis-Menten model;</p>b<p>WT = 100; ^c k_obs and K could not be determined in a reliable manner, since the substrate did not reach the range of saturating levels.</p

Close-up views of the active site cleft in the nucleotide-free and bound structures of the DNM1L GG construct in stereo.

<p>(<b>A</b>) The nucleotide-free form with the most relevant residue side chains of the five GTP binding stretches and citrate (FLC, yellow) displayed as stick models. Electron density of a 2F_o-F_c map is shown in grey and contoured at 1σ. The red sphere designates the catalytic water (<b>C</b>). (<b>B</b>) GMP-PNP complex of the DNM1L GG construct. The nucleotide is shown as stick model, while the red spheres represent water molecules, such as the bridging water (<b>B</b>) and one, which binds to the α-phosphate. The electron density of a 2F_o-F_c map is shown in grey and contoured at 1σ, surrounding the nucleotide and relevant parts of the structure with significant conformational changes with respect to the nucleotide-free form.</p

Detailed views of the GTP-binding elements switch I, switch II, G4 and G5 of DNM1L.

<p>(<b>A</b>) The canonical Mg²⁺ site between O1β and the O2γ is not occupied in the GMP-PNP-DNM1L structure (green). Also, no significant positional shift of Thr59 from the switch I loop takes place between apo- (grey) and nucleotide form (green). The unnatural N3β atom may favour the γ-phosphate conformation rotated by about 60° with respect to the transition state of GTP, shifting it about 2.5 Å away from the catalytic water. Only the nucleotide-free form exhibits the catalytic water molecule (C, grey) bound at the Thr59 carbonyl O (3.15 Å) and connected to switch II via the NH of Gly149 (2.87 Å). The bridging H₂O (B, red) is only present in the nucleotide complex, bound to the Gly149 carbonyl O (3.21 Å). Upon GMP-PNP binding, the Arg53 side chain moves out of the active site, making room for an H₂O, which binds O2 of the α-phosphate. (<b>B</b>) The ribose of GMP-PNP forms bonds with the ether oxygen O4 to the Nζ of Lys216 (3.16 Å), and with the hydroxyl group of O2 to an H₂O (3.25 Å), which is bonded to the carbonyl O of Arg247 (2.45 Å) and the Ser40 Oγ (3.01 Å). Another bond is formed by the ribose O2 to Gln249 NH (2.79 Å). (<b>C</b>) The Lys216 side chain, depicted as thin stick model for clarity, covers the aromatic rings of the guanine part, while the Asp218 carboxylate binds the amino N2 (2.86 Å) and the N1 (3.12 Å). A further interaction from the Asn246 Oδ1 to the N7 (3.54 Å) might be mediated by an unresolved H₂O, which could be bound to the carbonyl O of Gly37, as seen in other dynamin-nucleotide complexes.</p