Allosteric activation of VCP, a AAA unfoldase, by small molecule mimicry.

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme activity, activator binding must be permissive to different conformational states needed for enzyme function. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, a AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP Activator 1 (VA1), a compound that dose-dependently stimulates VCP ATPase activity up to ∼3-fold. Our cryo-EM studies resulted in structures (∼2.9-3.5 Å-resolution) of VCP in apo and ADP-bound states, and revealed VA1 binding an allosteric pocket near the C-terminus in both states. Engineered mutations in the VA1 binding site confer resistance to VA1, and furthermore, modulate VCP activity to a similar level as VA1-mediated activation. The VA1 binding site can alternatively be occupied by a phenylalanine residue in the VCP C-terminal tail, a motif that is post-translationally modified and interacts with cofactors. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry. Significance: The loss of function of valosin-containing protein (VCP/p97), a mechanoenzyme from the AAA superfamily that hydrolyzes ATP and uses the released energy to extract or unfold substrate proteins, is linked to protein aggregation-based disorders. However, druggable allosteric sites to activate VCP, or any AAA mechanoenzyme, have not been identified. Here, we report cryo-EM structures of VCP in two states in complex with VA1, a compound we identified that dose-dependently stimulates VCP's ATP hydrolysis activity. The VA1 binding site can also be occupied by a phenylalanine residue in the VCP C-terminal tail, suggesting that VA1 acts through mimicry of this interaction. Our study reveals a druggable allosteric site and a mechanism of enzyme regulation.

Half-life systematics across the N=126 shell closure: role of first-forbidden transitions in the ß decay of heavy neutron-rich nuclei.

This Letter reports on a systematic study of ß-decay half-lives of neutron-rich nuclei around doubly magic (208)Pb. The lifetimes of the 126-neutron shell isotone (204)Pt and the neighboring (200-202)Ir, (203)Pt, (204)Au are presented together with other 19 half-lives measured during the "stopped beam" campaign of the rare isotope investigations at GSI collaboration. The results constrain the main nuclear theories used in calculations of r-process nucleosynthesis. Predictions based on a statistical macroscopic description of the first-forbidden ß strength reveal significant deviations for most of the nuclei with N<126. In contrast, theories including a fully microscopic treatment of allowed and first-forbidden transitions reproduce more satisfactorily the trend in the measured half-lives for the nuclei in this region, where the r-process pathway passes through during ß decay back to stability.

Using radiative transfer equation to model absorption by thin Cu(In,Ga)Se2 solar cells with Lambertian back reflector.

We investigate the optical absorption in a thin Cu(In,Ga)Se(2) solar cell with a Lambertian white paint beneath a transparent back contact. Although this configuration has been proposed more than 30 years ago, it turns out that rigorous simulation of Maxwell's equations demand powerful numerical calculations. This type of approach is time consuming and does not provide a physical insight in the absorption mechanisms. Here, we use the radiative transfer equation to deal with multiple scattering of the diffuse part of the light. The collimated part is treated accounting for wave effects. Our model is in good agreement with optical measurements.

New isomers in the full seniority scheme of neutron-rich lead isotopes: the role of effective three-body forces.

The neutron-rich lead isotopes, up to (216)Pb, have been studied for the first time, exploiting the fragmentation of a primary uranium beam at the FRS-RISING setup at GSI. The observed isomeric states exhibit electromagnetic transition strengths which deviate from state-of-the-art shell-model calculations. It is shown that their complete description demands the introduction of effective three-body interactions and two-body transition operators in the conventional neutron valence space beyond (208)Pb.

Enhanced scattering and absorption due to the presence of a particle close to an interface.

We study the influence of the presence of an interface on the scattering by a Rayleigh scatterer. The influence of an interface on the spontaneous emission has been known for many years. Here, we study the influence on the extinction cross-section and absorption cross-section. We provide a detailed analysis of interference and near-field effects. We show that the presence of a Rayleigh scatterer may enhance the specular reflection or specular transmission under certain conditions. Finally, we analyze the enhancement of absorption in the bulk in the presence of a small scatterer.

Superallowed Gamow-Teller decay of the doubly magic nucleus 100Sn.

The shell structure of atomic nuclei is associated with 'magic numbers' and originates in the nearly independent motion of neutrons and protons in a mean potential generated by all nucleons. During ß(+)-decay, a proton transforms into a neutron in a previously not fully occupied orbital, emitting a positron-neutrino pair with either parallel or antiparallel spins, in a Gamow-Teller or Fermi transition, respectively. The transition probability, or strength, of a Gamow-Teller transition depends sensitively on the underlying shell structure and is usually distributed among many states in the neighbouring nucleus. Here we report measurements of the half-life and decay energy for the decay of (100)Sn, the heaviest doubly magic nucleus with equal numbers of protons and neutrons. In the ß-decay of (100)Sn, a large fraction of the strength is observable because of the large decay energy. We determine the largest Gamow-Teller strength so far measured in allowed nuclear ß-decay, establishing the 'superallowed' nature of this Gamow-Teller transition. The large strength and the low-energy states in the daughter nucleus, (100)In, are well reproduced by modern, large-scale shell model calculations.

Determination of the natural radioactivity levels in north west of Dukhan, Qatar using high-resolution gamma-ray spectrometry.

This study is aimed at the determination of the activity concentrations of naturally occuring and technologically enhanced levels of radiation in 34 representative soil samples that have been collected from an inshore oil field area which was found to have, in a previous study, the highest observed value of 226Ra concentration among 129 soil samples. The activity concentrations of 238U and 226Ra have been inferred from gamma-ray transitions associated with their decay progenies and measured using a hyper-pure germanium detector. Details of the sample preparation and the gamma-ray spectroscopic analysis techniques are presented, together with the values of the activity concentrations associated with the naturally occuring radionuclide chains for all the samples collected from NW Dukhan. Discrete-line, gamma-ray energy transitions from spectral lines ranging in energy from ∼100 keV up to 2.6 MeV have been associated with characteristic decays of the various decay products within the 235.8U and 232Th radioactive decay chains. These data have been analyzed, under the assumption of secular equilibrium for the U and Th decay chains. Details of the sample preparation and the gamma-ray spectroscopic analysis techniques are presented. The weighted mean value of the activity concentrations of 226Ra in one of the samples was found to be around a factor of 2 higher than the values obtained in the previous study and approximately a factor of 10 higher than the accepted worldwide average value of 35 Bq/kg. The weighted mean values of the activity concentrations of 232Th and 40K were also deduced and found to be within the worldwide average values of 30 and 400 Bq/kg, respectively. Our previous study reported a value of 201.9±1.5Stat.±13Syst.Bq/kg for 226Ra in one sample and further investigation in the current work determined a measured value for 226Ra of 342.00±1.9Stat.±25Syst.Bq/kg in a sample taken from the same locality. This is significantly higher than all the other investigated soil samples in the current and previous works. Notably, the Th levels in the same sample are within the worldwide average expectations, implying that the increased 226Ra concentration arises from TENORM processes.

The Philadelphia variant of galactokinase: impaired [1-14C]galactose oxidation by intact erythrocytes.

Probands with the Philadelphia variant of galactokinase (GALKP) are black people who exhibit reduced galactokinase (GALK) activity in their red blood cells (RBC), but normal activity in their white blood cells (WBC). This reduced RBC GALK was demonstrated in disrupted erythrocytes. To investigate the possibility of a missing cofactor in hemolysates from individuals with GALKP phenotype, we compared [1-14C]galactose oxidation by intact erythrocytes, with the direct GALK assay in disrupted erythrocytes. The rate of [1-14C]glucose oxidation was also measured in order to differentiate an impaired galactose metabolism from a defect further along the pentose phosphate pathway. A good correlation (p less than 0.001) was found between the direct GALK assay and [1-14C]galactose oxidation in control subjects, which indicates that this method can be used effectively for the detection of GALK defects. This was further supported by studies on samples from heterozygotes and homozygotes for the GALKG deficient gene. For all the probands with a GALKP phenotype, diminished CO2 production from galactose was observed in the absence of impaired glucose metabolism. This allowed us to confirm the existence of a GALK deficiency in intact erythrocytes due to the GALKP variant. Further studies of RBC GALK catalytic properties are needed to investigate the molecular basis of this GALK deficiency.

Use of platelets, mononuclear and polymorphonuclear cells in the diagnosis of glycogen storage disease type VI.

We determined glycogen concentration and phosphorylase 'a+b' and phosphorylase a activities in platelets, mononuclear and polymorphonuclear cells from control subjects and patients with phosphorylase kinase deficiency (glycogen storage disease IX) and liver phosphorylase deficiency (glycogen storage disease VI). Variations according to cellular type and to subjects' age (1-40 years) were established. Variable glycogen overloading was found in all our patients. Glycogen storage disease (GSD) VI was characterized by a diminished total phosphorylase activity with a low or normal a/(a+b) ratio of phosphorylase activity. GSD IX was characterized by a very low residual activity of phosphorylase a with an 'a+b' activity low or normal.

High-pressure stopped-flow fluorometry at subzero temperatures: application to kinetics of the binding of NADH to liver alcohol dehydrogenase.

A stopped-flow apparatus operating in fluorescence mode over temperature and pressure ranges of +30 to -30 degrees C and 10(-3) to 2 kbar, respectively, is described. The system was interfaced on a special spectrofluorometer. Its general design is an improvement of the previous instrument (C. Balny, J. L. Saldana, and N. Dahan, (1984) Anal. Biochem. 139, 178-189) in that the observation chamber and the driving mechanism have been modified. The application of the method to kinetics of the binding of NADH to horse liver alcohol dehydrogenase at subzero temperatures and as a function of hydrostatic pressure is described.

Estrone and dehydroepiandrosterone sulfatase activities in guinea-pig uterus and liver: estrogenic effect of estrone sulfate.

Estrone and dehydroepiandrosterone (DHA) sulfatase activities were studied in the uterus and liver of female guinea-pigs (albino variety). The two activities were found in particulates, with the highest specific activity in microsomes. The effects of pH, buffers, temperature and the non-competitive inhibition of DHA sulfate on estrone sulfatase provided arguments for the existence of two distinct sulfatases. However, acrylamide gel electrophoresis of the solubilized microsome sulfatases gave a single peak for the two activities. In the uterus, the apparent Km of estrone and DHA sulfatases were 26.4 and 15.6 microM. Solubilized microsomal estrone sulfatase was inhibited by unconjugated steroids. The apparent Km of estrone sulfatase in liver was 10.7 microM. Estrone and DHA sulfatase activities were consistently lower in liver than in uterus and no DHA sulfatase activity was detected in fetal liver. In the uterus, the same sulfatase activities were found in female fetuses, castrated or mature females. Estrone sulfatase was significantly increased in the uterus of pregnant females (60-65 days gestation). Estrone sulfate was injected in vivo into mature castrated females. A significant increase in uterine weight and in uterine progesterone receptors was observed. The cytosol progesterone receptors were characterized by their Kd (1.40 nM) and by sucrose density gradient. It is concluded that the variations of estrone sulfatase activity in target tissues like the uterus may control the intracellular levels of biologically active estrogens.

High-pressure stopped-flow spectrometry at low temperatures.

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.

Estrone and dehydroepiandrosterone sulfatase activities and plasma estrone sulfate levels in human breast carcinoma.

The activity of the two membrane-bound sulfatases, estrone and dehydroepiandrosterone sulfatases, are reported in human breast carcinoma tissues. In 21 tested tumors (12 from post-menopausal women and 9 from nonmenopausal women), the two sulfatases were consistently present. The apparent Km values for estrone and dehydroepiandrosterone sulfatases were, respectively, 6.8 and 14.9 microM. In terms of maximal velocity, the sulfatase activities are not correlated to the estrogen or progesterone receptor status of the tumors or to the hormonal status of the donors. It may be concluded that these two activities are not hormone dependent. Estrone sulfate, the substrate of estrone sulfatase, has been measured in plasma of postmenopausal women. The mean levels (nmol/liter) of plasma estrone sulfate were compared in post-menopausal women with (n = 51) or without (n = 39) breast cancer. For the first age group (48 to 55 years old), no statistically significant difference in these levels was observed [1.91 +/- 1.06 versus 1.50 +/- 1.04 (mean +/- t0.95 (Formula: see text) S.E.)]. For the two other age groups (56 to 65 and 66 to 80 years of age), the differences were statistically significant [1.46 +/- 0.43 versus 0.77 +/- 0.21 (p less than 0.02) and 1.77 +/- 0.53 versus 0.81 +/- 0.22 (p less than 0.01)]. The usefulness of plasma estrone 3-sulfate levels as an indicator of the real estrogen status of postmenopausal women is discussed.