Oligomerization of G protein-coupled receptors: past, present, and future

G protein-coupled receptor (GPCR)-mediated signal transduction has been studied for more than a century. Despite the intense focus on this class of proteins, a molecular understanding of what constitutes the functional form of the receptor is still uncertain. GPCRs have traditionally been conceptualized as monomeric proteins, and this view has changed little over the years until relatively recently. Recent biochemical and biophysical studies have challenged this traditional concept, and point instead to a mechanistic view of signal transduction wherein the receptor functions as an oligomer. Cooperative interactions within such an oligomeric array may be critical for the propagation of an external signal across the cell membrane and to the G protein, and may therefore underlie the mechanistic basis of signaling.     

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis,Genome Evolution and Niche Adaptation,and Infection Detection

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V² values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates.     

Covalent modification of Alzheimer's amyloid beta-peptide in formic acid solutions.

beta-peptide is a normal component of amyloid deposits in Alzheimer's disease. In aqueous solution, beta-peptide is extremely insoluble and rapidly aggregates forming oligomeric beta-sheet structures that eventually precipitate from solution. Presumably, this process is related to the production of amyloid deposits in Alzheimer's disease. Formic acid is commonly used to dissolve the beta-peptides and prevent aggregation in biological and biophysical studies. However, a side-reaction which covalently modifies beta-peptide is encountered with formic acid. In this report, fast atom bombardment mass spectrometry and tandem mass spectrometry demonstrate that both Ser8 and Ser26 become O-formylated in 70% aqueous formic acid solutions. The implications of this O-formylation upon the aggregational properties of beta-peptide are discussed.     

Maternal Plasma and Amniotic Fluid Sphingolipids Profiling in Fetal Down Syndrome

IntroductionSphingolipids can be potentially involved in the formation of the central and peripheral nervous systems, which are particularly connected with the pathogenesis of Down syndrome. The aim of the study was to determine the concentration of selected sphingolipids in the plasma and amniotic fluid of pregnant patients with fetal Down syndrome.ResultsWe showed a significant increase in the concentration of 2 ceramides, C22-Cer and C24:1-Cer, in the plasma of women with fetal Down syndrome. Furthermore we showed a decrease in the concentration of 7 ceramides—C16-Cer, C18-Cer, C18:1-Cer, C20-Cer, C22-Cer, C24:1-Cer, and C24-Cer—in the amniotic fluid of women with fetal Down syndrome. We created ROC curves for all significant sphingolipids in maternal plasma, which set the threshold values and allowed for predicting the likelihood of Down syndrome in the fetus with specific sensitivity and specificity. We demonstrated a significantly higher risk of Down syndrome when the plasma concentration of C22-Cer > 12.66 ng/100ul (sens. 0.9, sp. 0.79, P value = 0.0007) and C24:1-Cer > 33,19 ng/100ul (sens. 0.6, sp. 0.86, P value = 0.0194).ConclusionOn the basis of our findings, it seems that the sphingolipids may play a role in the pathogenesis of Down syndrome. Defining their potential as biochemical markers of Down syndrome requires further investigation on a larger group of patients.     

Aspergillus Collagen-Like Genes (acl): Identification,Sequence Polymorphism,and Assessment for PCR-Based Pathogen Detection

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.     

Secondary structure of alpha-synuclein oligomers: characterization by raman and atomic force microscopy

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.     

A novel retinoid binding property of human annexin A6

Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.