New perspectives for pharmacological chaperoning treatment in methylmalonic aciduria cblB type

Methylmalonic aciduria cblB type (MMA cblB) is caused by the impairment of ATP:cob(I)alamin adenosyltransferase (ATR), the enzyme responsible for the synthesis of adenosylcobalamin (AdoCbl) from cob(I)alamin. No definitive treatment is available for patients with this condition and novel therapeutic strategies are therefore much needed. Recently, we described a proof-of-concept regarding the use of pharmacological chaperones as a treatment. This work describes the effect of two potential pharmacological chaperones - compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) and compound VI (4-(4-(4-fluorophenyl)-5-methyl-1H-pyrazol-3-yl)benzene-1,3-diol) - on six ATR mutants, including the most common, p.Arg186Trp. Comprehensive functional analysis identified destabilizing (p.Arg186Gln, p.Arg190Cys, p.Arg190His, p.Arg191Gln and p.Glu193Lys) and oligomerization (p.Arg186Trp and p.Arg191Gln) mutations. In a cellular model overexpressing the destabilizing/oligomerization mutations, compounds V and VI had a positive effect on the stability and activity of all ATR variants. When provided in combination with hydroxocobalamin a more positive effect was obtained than with the compounds alone, even in mutations previously described as B12 non-responsive. In addition, a normal oligomerization profile was recovered after treatment of the p.Arg186Trp mutant with both compounds. These promising results confirm MMA cblB type as a conformational disorder and hence, pharmacological chaperones as a new therapeutic option alone or in combination with hydroxocobalamin for many patients with MMA cblBThis work was supported by Instituto de Salud Carlos III and (grant PI13/01239) plus grants from the Fundación Isabel Gemio and Obra Social de La Caixa to BP; the Research Council of Norway [nr. 185181 to AM], The KG Jebsen Foundation, and NovoSeeds (Novo Nordisk). AG was supported by a Ramón y Cajal grant from the Ministerio de Ciencia y Tecnología. This work was supported also by the European Regional Development Fund (PI13/01239

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Conformational studies of peptides representing a segment of TM7 from H+-VO-ATPase in SDS micelles

The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an α-helical conformation or aggregated β-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.e., MTM7). It is concluded that the bulky, positively charged arginine residue located in the center of both peptides has a destabilizing effect on the helical conformation of the SDS-solubilized peptides, leading to β-sheet formation and subsequent aggregation

The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.This work was supported by grants from the Norwegian Cancer Society (to ØH), Junta de Andalucía, grant CVI-02483 (to JMSR), The Research Council of Norway (grant 185181 to A.M.), the Western Norway Health Authorities (grant 911618 to A.M.) and The Kristian Gerhard Jebsen Foundation (to AM)

In thrombin stimulated human platelets Citalopram, Promethazine, Risperidone, and Ziprasidone, but not Diazepam, may exert their pharmacological effects also through intercalation in membrane phospholipids in a receptor-independent manner

Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs’ intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A2 (PLA2) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA2 activity, had no effects on PPI cycle, and caused no change in mean molecular area of phospholipid monolayers. The remaining psychotropic drugs affected these parameters in different ways and levels of potency suggesting that they act by being intercalated between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for phospholipid-hydrolyzing enzymes (PLA2 and Phospholipase C). We show that several psychotropic drugs can also have other cellular effects than receptor antagonism. These effects may be implicated in the psychotropic effects of the drugs and/or their side effects

Linking genotypes database with locus-specific database and genotype-phenotype correlation in phenylketonuria.

The wide range of metabolic phenotypes in phenylketonuria is due to a large number of variants causing variable impairment in phenylalanine hydroxylase function. A total of 834 phenylalanine hydroxylase gene variants from the locus-specific database PAHvdb and genotypes of 4181 phenylketonuria patients from the BIOPKU database were characterized using FoldX, SIFT Blink, Polyphen-2 and SNPs3D algorithms. Obtained data was correlated with residual enzyme activity, patients' phenotype and tetrahydrobiopterin responsiveness. A descriptive analysis of both databases was compiled and an interactive viewer in PAHvdb database was implemented for structure visualization of missense variants. We found a quantitative relationship between phenylalanine hydroxylase protein stability and enzyme activity (rs=0.479), between protein stability and allelic phenotype (rs=-0.458), as well as between enzyme activity and allelic phenotype (rs=0.799). Enzyme stability algorithms (FoldX and SNPs3D), allelic phenotype and enzyme activity were most powerful to predict patients' phenotype and tetrahydrobiopterin response. Phenotype prediction was most accurate in deleterious genotypes (≈100%), followed by homozygous (92.9%), hemizygous (94.8%), and compound heterozygous genotypes (77.9%), while tetrahydrobiopterin response was correctly predicted in 71.0% of all cases. To our knowledge this is the largest study using algorithms for the prediction of patients' phenotype and tetrahydrobiopterin responsiveness in phenylketonuria patients, using data from the locus-specific and genotypes database.European Journal of Human Genetics advance online publication, 18 June 2014; doi:10.1038/ejhg.2014.114

Conformational stabilization as a strategy to prevent nucleophosmin mislocalization in leukemia

Nucleophosmin (NPM) is a nucleolar protein involved in ribosome assembly and cell homeostasis. Mutations in the C-terminal domain of NPM that impair native folding and localization are associated with acute myeloid leukemia (AML). We have performed a high-throughput screening searching for compounds that stabilize the C-terminal domain. We identified three hit compounds which show the ability to increase the thermal stability of both the C-terminal domain as well as full-length NPM. The best hit also seemed to favor folding of an AML-like mutant. Computational pocket identification and molecular docking support a stabilization mechanism based on binding of the phenyl/benzene group of the compounds to a particular hydrophobic pocket and additional polar interactions with solvent-accessible residues. Since these results indicate a chaperoning potential of our candidate hits, we tested their effect on the subcellular localization of AML-like mutants. Two compounds partially alleviated the aggregation and restored nucleolar localization of misfolded mutants. The identified hits appear promising as pharmacological chaperones aimed at therapies for AML based on conformational stabilization of NPM

Conformational stabilization as a strategy to prevent nucleophosmin mislocalization in leukemia

Abstract Nucleophosmin (NPM) is a nucleolar protein involved in ribosome assembly and cell homeostasis. Mutations in the C-terminal domain of NPM that impair native folding and localization are associated with acute myeloid leukemia (AML). We have performed a high-throughput screening searching for compounds that stabilize the C-terminal domain. We identified three hit compounds which show the ability to increase the thermal stability of both the C-terminal domain as well as full-length NPM. The best hit also seemed to favor folding of an AML-like mutant. Computational pocket identification and molecular docking support a stabilization mechanism based on binding of the phenyl/benzene group of the compounds to a particular hydrophobic pocket and additional polar interactions with solvent-accessible residues. Since these results indicate a chaperoning potential of our candidate hits, we tested their effect on the subcellular localization of AML-like mutants. Two compounds partially alleviated the aggregation and restored nucleolar localization of misfolded mutants. The identified hits appear promising as pharmacological chaperones aimed at therapies for AML based on conformational stabilization of NPM

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization

ABOUT THE TEST REPORT AND USE OF THE DATA The test data contained in this report are a tabulation of the results of a series of tests. Due to the restricted format of these pages, only a limited amount of data and not all of the tractor specifications are included. The full OECD report contains usually about 30 pages of data and specifications. The test data were obtained for each tractor under similar conditions and therefore, provide a means of comparison of performance based on a limited set of reported data. EXPLANATION OF THE TEST PROCEDURES Purpose The purpose of the tests in this booklet, and available test reports is to provide users with data for comparisons of performance among tractor models. General Tractors are tested at the University of Nebraska according to test procedures of the OECD (Organization of Economic Cooperation and Development), the SAE (Society of Automotive Engineers) International and the ASABE (American Society of Agricultural and Biological Engineers). The three codes are technically equivalent, but do differ slightly. For the past 10 years, the majority of tests have been performed according to the OECD codes. The manufacturer selects the tractor to be tested from its production line, provides the specifications, and certifies that the tractor is a stock model. Each tractor is equipped with the common energy consuming accessories (power steering, PTO, implement lifts, etc.). Any power consuming accessory may be disconnected when the means for doing so can be reached from the operator position. A manufacturer\u27s representative is present during the tests to as certain that the tractor gives its optimum performance. Weight can be added to the tractor to improve drawbar performance in certain tests. Static tire loads and inflation pressures must conform to the specifications of the Tire and Rim Association or to weight limits set by the manufacturer. Specifications All manufacturers provide the Laboratory with detailed specifications which are required for the tests. The Nebraska Tractor Test report provides only a limited amount of data due to space constraints. Preparation for Test The tractor is required to have been limbered up by the manufacturer for a sufficient number of hours; if this was not done, this limber- up is performed at the Tractor Test Lab. Adjustments are permitted during this period. After the start of the official test, no adjustments can be made. Any adjustments. repairs, alterations or replacements are mentioned in the final Nebraska Tractor Test report. At this time, instrumentation for measuring engine rpm, fan speed, temperatures and pressures is installed on the tractor. The tractor is also provided with connections to the Lab\u27s fuel supply. PTO Performance The tractor PTO is connected to a dynamometer, which is a device for putting a load on the tractor and measuring the power generated by the tractor. During the preliminary runs, the manufacturer is allowed to make some adjustments to optimize the performance. These adjustments, which include the injection pump volume and timing and the high idle set within the specified range, will remain during the whole test program and must be settings guaranteed by the manufacturer. The tests are performed while maintaining an ambient temperature of 75°F (24°C) and at a barometer reading above 28.5 inches Hg (96.6 kPa). Data are taken at intervals after the tractor performance has stabilized. Inlet fuel temperatures are also maintained at a predetermined level. The throttle being set for maximum no-load rpm (High Idle), an increasing load is applied to the PTO by the dynamometer along the operating curve of the engine. The full test report will show the torque, rpm, power and fuel consumption data obtained at Rated Engine speed, Standard PTO speed (either 1000 or 540 rpm), the maximum power on the curve and the torque rise. Drawbar Performance Tests are performed in all gears between one gear below the one at Which 15% slip occurs and a maximum speed of 10 mph (16.1 km/h). In each gear, the throttle is set for maximum speed (High Idle) and the drawbar load increased until maximum drawbar power is obtained. The drawbar load is created by towing load units behind the test-and- measurement vehicle which, itself, is hitched to the tested tractor\u27s drawbar. For each load, measurements and calculations are made to determine drawbar pull, speed, drawbar power, slip and fuel consumption. All measurements are recorded at intervals after the tractor\u27s condition is stabilized. No operational limits set by the manufacturer can be exceeded. A second test series investigates the part loads at 75% and 50% of the drawbar load at Rated Engine Speed in a gear close to 4.6 mph (7.5 km/h) and in the gear where maximum drawbar power was obtained. Sound Measurement Sound measurement is made on the test track in two locations-at the driver\u27s ear and in a location representing bystander noise. The tests at the driver\u27s ear are performed in several gears and under a number of conditions, but only the maximum level is reported. The bystander sound test is performed with the microphone located at 25 ft (7.5 m) from the centerline of the tractor which is accelerating from a lower speed to full speed in its top gear. The OECD procedure differs. The SAE/ASABE procedures and only the numbers for the same gears and same load conditions can be compared. The SAE/ASABE procedure measures sound in only one gear under different load conditions, whereas the GECD procedure measures sound in different gears between High Idle and Rated Engine speed. For tractors with Mechanical Front Wheel Drive, operator- ear measurements are made with the front-wheel drive engaged and disengaged. Hydraulic Lift Capacity and Flow Hydraulic lift capacity is measured in a special test stand. A frame is fitted to the three-point hitch lift links. Measurements of lift capacity are taken at the hitch points and at a point 24 (610 mm) behind the hitch points when the lower links are horizontal. The load is applied with a hydraulic cylinder and the arms move stepwise through the lift range. The number which is reported is 90% of the load which can be carried throughout the lift range. The booklet reports the lift capacity at 24 (610 mm) behind the hitch points. A second test determines the pressure/flow relationship and performance of the hydraulic system for supplying power to external hydraulic cylinders or hydraulic motors. The Nebraska report provides data on delivery rate, pressure and available powe