Structural and thermodynamic bases for the design of pure prolactin receptor antagonists: X-ray structure of Del1-9-G129R-hPRL

Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly(129) mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly(129) mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL.     

Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B'-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.     

Monohydroxylated poly(3-hydroxyoctanoate) oligomers and its functionalized derivatives used as macroinitiators in the synthesis of degradable diblock copolyesters

The presence of a hydroxyl group at the end of poly(3-hydroxyoctanoate) oligomers, noted PHO oligomers, is required to prepare diblock copolymers with improved properties by ring-opening polymerization of cyclic monomer as epsilon-caprolactone. Several chemical methods such as basic hydrolysis, acid-catalyzed reaction with APTS, and methanolysis were used to prepare well-defined low molar masses PHO oligomers. The methanolysis reaction was allowed to proceed for 10-60 min to produce PHO oligomers with Mn values ranging from 20,000 to 800 g mol-1 with low polydispersity index. Detailed analysis of the MALDI-TOF mass spectra of the obtained oligomers has revealed the presence of linear structures bearing methyl ester on one side and hydroxyl end group on the other side. The same procedure was applied to poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate), PHOU, a poly(3-hydroxyalkanoate) containing unsaturated units in its side chains. These oligomers were further used to initiate the polymerization of epsilon-caprolactone by varying the PHO (or PHOU) and PCL lengths. By copolymerization with epsilon-caprolactone, the properties of PHO or PHOU have been improved. The crystallinity of the obtained copolymers was modified by controlling the length of the two different blocks. The unsaturations in the side chains of the PHOU block were oxidized in acid carboxylic functions to obtain a novel artificial biopolyester. Moreover, degradation was followed to study the influence of carboxylic groups on the hydrolysis of the copolymers.     

Modification of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film by chemical graft copolymerization

The graft copolymerization of 2-hydroxyethylmethacrylate (HEMA) onto poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) films has been investigated. The graft copolymerization was conducted in aqueous media using benzoyl peroxide (BPO) as chemical initiator. PHBHV films were prepared by solvent casting. Different parameters affecting the graft yield were studied such as monomer concentration, initiator concentration, and reaction time. The extent of grafting has been modulated by the preparation conditions, in particular the concentration of HEMA. However, it is interesting to note that the initiator concentration had only a slight influence on the graft yield. Characterization of the grafted PHBHV films assumed that the graft copolymerization not only occurred on the film surface but also took place into the film bulk. Differential scanning calorimetry showed that crystallinity dramatically decreased with increasing graft yield, indicating that graft copolymerization hindered the crystallization process. Wettability has been obviously improved by grafting a hydrophilic monomer such as HEMA for high graft yield (>130%).     

LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling

The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.     

Detection of a Cis [corrected] eQTL controlling BCMO1 gene expression leads to the identification of a QTG for chicken breast meat color

Classical quantitative trait loci (QTL) analysis and gene expression QTL (eQTL) were combined to identify the causal gene (or QTG) underlying a highly significant QTL controlling the variation of breast meat color in a F2 cross between divergent high-growth (HG) and low-growth (LG) chicken lines. Within this meat quality QTL, BCMO1 (Accession number GenBank: AJ271386), encoding the β-carotene 15, 15'-monooxygenase, a key enzyme in the conversion of β-carotene into colorless retinal, was a good functional candidate. Analysis of the abundance of BCMO1 mRNA in breast muscle of the HG x LG F2 population allowed for the identification of a strong cis eQTL. Moreover, reevaluation of the color QTL taking BCMO1 mRNA levels as a covariate indicated that BCMO1 mRNA levels entirely explained the variations in meat color. Two fully-linked single nucleotide polymorphisms (SNP) located within the proximal promoter of BCMO1 gene were identified. Haplotype substitution resulted in a marked difference in BCMO1 promoter activity in vitro. The association study in the F2 population revealed a three-fold difference in BCMO1 expression leading to a difference of 1 standard deviation in yellow color between the homozygous birds at this haplotype. This difference in meat yellow color was fully consistent with the difference in carotenoid content (i.e. lutein and zeaxanthin) evidenced between the two alternative haplotypes. A significant association between the haplotype, the level of BCMO1 expression and the yellow color of the meat was also recovered in an unrelated commercial broiler population. The mutation could be of economic importance for poultry production by making possible a gene-assisted selection for color, a determining aspect of meat quality. Moreover, this natural genetic diversity constitutes a new model for the study of β-carotene metabolism which may act upon diverse biological processes as precursor of the vitamin A.     

Detection of a Cis eQTL Controlling BMCO1 Gene Expression Leads to the Identification of a QTG for Chicken Breast Meat Color

Classical quantitative trait loci (QTL) analysis and gene expression QTL (eQTL) were combined to identify the causal gene (or QTG) underlying a highly significant QTL controlling the variation of breast meat color in a F2 cross between divergent high-growth (HG) and low-growth (LG) chicken lines. Within this meat quality QTL, BCMO1 (Accession number GenBank: {"type":"entrez-nucleotide","attrs":{"text":"AJ271386","term_id":"7799040","term_text":"AJ271386"}}AJ271386), encoding the β-carotene 15, 15′-monooxygenase, a key enzyme in the conversion of β-carotene into colorless retinal, was a good functional candidate. Analysis of the abundance of BCMO1 mRNA in breast muscle of the HG x LG F2 population allowed for the identification of a strong cis eQTL. Moreover, reevaluation of the color QTL taking BCMO1 mRNA levels as a covariate indicated that BCMO1 mRNA levels entirely explained the variations in meat color. Two fully-linked single nucleotide polymorphisms (SNP) located within the proximal promoter of BCMO1 gene were identified. Haplotype substitution resulted in a marked difference in BCMO1 promoter activity in vitro. The association study in the F2 population revealed a three-fold difference in BCMO1 expression leading to a difference of 1 standard deviation in yellow color between the homozygous birds at this haplotype. This difference in meat yellow color was fully consistent with the difference in carotenoid content (i.e. lutein and zeaxanthin) evidenced between the two alternative haplotypes. A significant association between the haplotype, the level of BCMO1 expression and the yellow color of the meat was also recovered in an unrelated commercial broiler population. The mutation could be of economic importance for poultry production by making possible a gene-assisted selection for color, a determining aspect of meat quality. Moreover, this natural genetic diversity constitutes a new model for the study of β-carotene metabolism which may act upon diverse biological processes as precursor of the vitamin A.     

Identification of genetic markers for differentiation of Shiga toxin-producing, enteropathogenic, and avirulent strains of Escherichia coli O26

Shiga toxin-producing Escherichia coli (STEC) O26 is one of the top five enterohemorrhagic E. coli (EHEC) O groups most often associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. STEC O26 is considered to have evolved from enteropathogenic (EPEC) O26 strains through the acquisition of Shiga toxin (Stx)-encoding genes. Our PCR data identified several STEC-like strains expressing all features of STEC except Stx production and carrying remnants of Stx phages that were probably derivatives of EHEC O26. EHEC and EPEC O26 strains phenotypically resemble O26 EHEC-like and apathogenic E. coli O26 strains and are therefore undistinguishable by cultural methods. A clear discrimination between the different O26 groups is required for diagnostics in patients and for control of food safety. To develop an assay for specific detection of EHEC and EHEC-like O26 strains, we used a high-throughput PCR approach for selection of discriminative genetic markers among 33 tested genes mostly encoding type III secretion system effector proteins. The genes ECs1822, nleH1-2, nleA, nleC, nleH1-1, nleG, nleG2, nleG6-1, nleG6-2, espJ, espM2, nleG8-2, espG, ent (or espL2), nleB, nleE, efa1, and espB were detected at different frequencies in O26 EHEC, EHEC-like, and EPEC strains, indicating the possible role of these genes in virulence of human pathogenic O26 strains. The espK and espN genes were detected only in EHEC and EHEC-like O26 strains. espK was present in 99.14% of EHEC and 91.14% of EHEC-like O26 strains and was hence the best candidate as a genetic marker for characterizing these pathogroups. These data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. The results indicate that a combination of molecular detection tools for O26 wzx (wzx(O26)), eae-beta, stx, espK, and arcA genotyping is highly discriminative for clear identification of EHEC and EHEC-like E. coli O26 strains. This simple diagnostic test might be applicable in hospital service laboratories or public health laboratories to test strains isolated from stools of patients suffering from diarrhea.