Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

Roux-en-Y Gastric Bypass Alters Brain Activity in Regions that Underlie Reward and Taste Perception

Background

Roux-en-Y gastric bypass (RYGB) surgery is a very effective bariatric procedure to achieve significant and sustained weight loss, yet little is known about the procedure’s impact on the brain. This study examined the effects of RYGB on the brain’s response to the anticipation of highly palatable versus regular food.

Methods

High fat diet-induced obese rats underwent RYGB or sham operation and were then tested for conditioned place preference (CPP) for the bacon-paired chamber, relative to the chow-paired chamber. After CPP, animals were placed in either chamber without the food stimulus, and brain-glucose metabolism (BGluM) was measured using positron emission tomography (μPET).

Results

Bacon CPP was only observed in RYGB rats that had stable weight loss following surgery. BGluM assessment revealed that RYGB selectively activated regions of the right and midline cerebellum (Lob 8) involved in subjective processes related to reward or expectation. Also, bacon anticipation led to significant activation in the medial parabrachial nuclei (important in gustatory processing) and dorsomedial tegmental area (key to reward, motivation, cognition and addiction) in RYGB rats; and activation in the retrosplenial cortex (default mode network), and the primary visual cortex in control rats.

Conclusions

RYGB alters brain activity in areas involved in reward expectation and sensory (taste) processing when anticipating a palatable fatty food. Thus, RYGB may lead to changes in brain activity in regions that process reward and taste-related behaviors. Specific cerebellar regions with altered metabolism following RYGB may help identify novel therapeutic targets for treatment of obesity.     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

Alpha repeat proteins (αRep) as expression and crystallization helpers

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.     

Maturation of Human Herpesvirus 6A Glycoprotein O Requires Coexpression of Glycoprotein H and Glycoprotein L

Glycoprotein O (gO) is conserved among betaherpesviruses, but little is known about the maturation process of gO in human herpesvirus 6 (HHV-6). We found that HHV-6 gO maturation was accompanied by cleavage of its carboxyl terminus and required coexpression of gH and gL, which promoted the export of gO out of the endoplasmic reticulum (ER). Finally, we also found that gO was not required for HHV-6A growth in T cells.     

Effect of Cattle on Salmonella Carriage,Diversity and Antimicrobial Resistance in Free-Ranging Wild Boar (Sus scrofa) in Northeastern Spain

Salmonella is distributed worldwide and is a pathogen of economic and public health importance. As a multi-host pathogen with a long environmental persistence, it is a suitable model for the study of wildlife-livestock interactions. In this work, we aim to explore the spill-over of Salmonella between free-ranging wild boar and livestock in a protected natural area in NE Spain and the presence of antimicrobial resistance. Salmonella prevalence, serotypes and diversity were compared between wild boars, sympatric cattle and wild boars from cattle-free areas. The effect of age, sex, cattle presence and cattle herd size on Salmonella probability of infection in wild boars was explored by means of Generalized Linear Models and a model selection based on the Akaike’s Information Criterion. Prevalence was higher in wild boars co-habiting with cattle (35.67%, CI 95% 28.19–43.70) than in wild boar from cattle-free areas (17.54%, CI 95% 8.74–29.91). Probability of a wild boar being a Salmonella carrier increased with cattle herd size but decreased with the host age. Serotypes Meleagridis, Anatum and Othmarschen were isolated concurrently from cattle and sympatric wild boars. Apart from serotypes shared with cattle, wild boars appear to have their own serotypes, which are also found in wild boars from cattle-free areas (Enteritidis, Mikawasima, 4:b:- and 35:r:z35). Serotype richness (diversity) was higher in wild boars co-habiting with cattle, but evenness was not altered by the introduction of serotypes from cattle. The finding of a S. Mbandaka strain resistant to sulfamethoxazole, streptomycin and chloramphenicol and a S. Enteritidis strain resistant to ciprofloxacin and nalidixic acid in wild boars is cause for public health concern.     

Identification of a Potent Endothelium-Derived Angiogenic Factor

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up₄U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up₄U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up₄U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up₄U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up₄U after stimulation with shear stress. Mean total plasma Up₄U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L^-1). In conclusion, Up₄U is a novel strong human endothelium-derived angiogenic factor.