Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/β γ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.     

Indirect DNA readout by an H-NS related protein: structure of the DNA complex of the C-terminal domain of Ler

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.     

Mitochondrial inhibitory factor 1 (IF1) is present in human serum and is positively correlated with HDL-cholesterol

Background

Mitochondrial ATP synthase is expressed as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein component in High Density Lipoproteins (HDL). On hepatocytes, apoA-I binds to cell surface ATP synthase (namely ecto-F₁-ATPase) and stimulates its ATPase activity, generating extracellular ADP. This production of extracellular ADP activates a P2Y₁₃-mediated HDL endocytosis pathway. Conversely, exogenous IF1, classically known as a natural mitochondrial specific inhibitor of F₁-ATPase activity, inhibits ecto-F₁-ATPase activity and decreases HDL endocytosis by both human hepatocytes and perfused rat liver.

Methodology/Principal Findings

Since recent reports also described the presence of IF1 at the plasma membrane of different cell types, we investigated whether IF1 is present in the systemic circulation in humans. We first unambiguously detected IF1 in human serum by immunoprecipitation and mass spectrometry. We then set up a competitive ELISA assay in order to quantify its level in human serum. Analyses of IF1 levels in 100 normolipemic male subjects evidenced a normal distribution, with a median value of 0.49 µg/mL and a 95% confidence interval of 0.22–0.82 µg/mL. Correlations between IF1 levels and serum lipid levels demonstrated that serum IF1 levels are positively correlated with HDL-cholesterol and negatively with triglycerides (TG).

Conclusions/Significance

Altogether, these data support the view that, in humans, circulating IF1 might affect HDL levels by inhibiting hepatic HDL uptake and also impact TG metabolism.     

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71

Summary Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.     

Semi-automated recognition of protozoa by image analysis

A programme was created to semi-automatically analyse protozoal digitised images. Principal Component Analysis technique was used for species identification. After data collection and mathematical treatment, a three-dimensional representation was generated and several protozoa (Opercularia, Colpidium, Tetrahymena, Prorodon, Glaucoma and Trachelophyllum) species could be positively identified.     

Defining Body Fatness in Adolescents: A Proposal of the Afad-A Classification

Aims

Body mass index (BMI) shows several limitations as indicator of fatness. Using the International Obesity Task Force (IOTF) reference and the World Health Organization (WHO) standard 2007 on the same dataset yielded widely different rates. At higher levels, BMI and the BMI cut-offs may be help in informing a clinical judgement, but at levels near the norm additional criteria may be needed. This study compares the prevalence of overweight and obesity using IOTF and WHO-2007 references and interprets body composition by comparing measures of BMI and body fatness (fat mass index, FMI; and waist-to-height ratio, WHtR) among an adolescent population.

Methods and Results

A random sample (n = 1231) of adolescent population (12–17 years old) was interviewed. Weight, height, waist circumference, triceps and subscapular skinfolds were used to calculate BMI, FMI, and WHtR. The prevalence of overweight and obesity were 12.3% and 15.4% (WHO standards) and 18.6% and 6.1% (IOTF definition). Despite that IOTF cut-offs misclassified less often than WHO standards, BMI categories were combined with FMI and WHtR resulting in the Adiposity & Fat Distribution for adolescents (AFAD-A) classification, which identified the following groups normal-weight normal-fat (73.2%), normal-weight overfat (2.1%), overweight normal-fat (6.7%), overweight overfat (11.9%) and obesity (6.1%), and also classified overweight at risk and obese adolescents into type-I (9.5% and 1.3%, respectively) and type-II (2.3% and 4.9%, respectively) depending if they had or not abdominal fatness.

Conclusions

There are differences between IOTF and WHO-2007 international references and there is a misclassification when adiposity is considered. The BMI limitations, especially for overweight identification, could be reduced by adding an estimate of both adiposity (FMI) and fat distribution (WHtR). The AFAD-A classification could be useful in clinical and population health to identify overfat adolescent and those who have greater risk of developing weight-related cardiovascular diseases according to the BMI category.     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

Multiple models for Rosaceae genomics

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.     

Prevention of diabetes by manipulation of anti-IGRP autoimmunity: high efficiency of a low-affinity peptide

Antigen therapy may hold great promise for the prevention of autoimmunity; however, most clinical trials have failed, suggesting that the principles guiding the choice of treatment remain ill defined. Here, we examine the antidiabetogenic properties of altered peptide ligands of CD8+ T cells recognizing an epitope of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP206-214), a prevalent population of autoreactive T cells in autoimmune diabetes. We show that islet-associated CD8+ T cells in nonobese diabetic mice recognize numerous IGRP epitopes, and that these cells have a role in the outcome of protocols designed to induce IGRP206-214-specific tolerance. Ligands targeting IGRP206-214-reactive T cells prevented disease, but only at doses that spared low-avidity clonotypes. Notably, near complete depletion of the IGRP206-214-reactive T-cell pool enhanced the recruitment of subdominant specificities and did not blunt diabetogenesis. Thus, peptide therapy in autoimmunity is most effective under conditions that foster occupation of the target organ lymphocyte niche by nonpathogenic, low-avidity clonotypes.     

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.