Zwitterionic Character and Lipid Composition Determine the Behaviour of Glycosylphosphatidylinositol Fragments in Monolayers

Glycosylphosphatidylinositols (GPIs) are complex glycolipids found in free form or anchoring proteins to the outer leaflet of the cell membrane in eukaryotes. GPIs have been associated with the formation of lipid rafts and protein sorting on membranes. The presence of a conserved glycan core with cell-specific modifications together with lipid remodelling during biosynthesis suggest that the properties of the glycolipids are being fine-tuned. We synthesized a series of GPI fragments and evaluated the interactions and arrangement of these glycolipids in monolayers as a 2-D membrane model. GIXD and IRRAS analyses showed the need of N-acetylglucosamine deacetylation for the formation of hydrogen bonds to obtain highly structured domains in the monolayers and an effect of the unsaturated lipids in formation and localization of the glycolipids within or between membrane microdomains. These results contribute to understand the role of these glycolipids and their modifications in the organization of membranes

Synthetic phosphoethanolamine-modified oligosaccharides reveal the importance of glycan length and substitution in biofilm-inspired assemblies

Bacterial biofilm matrices are nanocomposites of proteins and polysaccharides with remarkable mechanical properties. Efforts understanding and tuning the protein component have been extensive, whereas the polysaccharide part remained mostly overlooked. The discovery of phosphoethanolamine (pEtN) modified cellulose in E. coli biofilms revealed that polysaccharide functionalization alters the biofilm properties. To date, the pattern of pEtN cellulose and its mode of interactions with proteins remains elusive. Herein, we report a model system based on synthetic epitomes to explore the role of pEtN in biofilm-inspired assemblies. Nine pEtN-modified oligosaccharides were synthesized with full control over the length, degree and pattern of pEtN substitution. The oligomers were co-assembled with a representative peptide, triggering the formation of fibers in a length dependent manner. We discovered that the pEtN pattern modulates the adhesion of biofilm-inspired matrices, while the peptide component controls its stiffness. Unnatural oligosaccharides tune or disrupt the assembly morphology, revealing interesting targets for polysaccharide engineering to develop tunable bio-inspired materials

Refined histopathological predictors of BRCA1 and BRCA2 mutation status: A large-scale analysis of breast cancer characteristics from the BCAC, CIMBA, and ENIGMA consortia

Introduction: The distribution of histopathological features of invasive breast tumors in BRCA1 or BRCA2 germline mutation carriers differs from that of individuals with no known mutation. Histopathological features thus have utility for mutation prediction, including statistical modeling to assess pathogenicity of BRCA1 or BRCA2 variants of uncertain clinical significance. We analyzed large pathology datasets accrued by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and the Breast Cancer Association Consortium (BCAC) to reassess histopathological predictors of BRCA1 and BRCA2 mutation status, and provide robust likelihood ratio (LR) estimates for statistical modeling. Methods: Selection criteria for study/center inclusion were estrogen receptor (ER) status or grade data available for invasive breast cancer diagnosed younger than 70 years. The dataset included 4,477 BRCA1 mutation carriers, 2,565 BRCA2 mutation carriers, and 47,565 BCAC breast cancer cases. Country-stratified estimates of the

Identification of four novel susceptibility loci for oestrogen receptor negative breast cancer

Common variants in 94 loci have been associated with breast cancer including 15 loci with genome-wide significant associations (P<5 × 10−8) with oestrogen receptor (ER)-negative breast cancer and BRCA1-associated breast cancer risk. In this study, to identify new ER-negative susceptibility loci, we performed a meta-analysis of 11 genome-wide association studies (GWAS) consisting of 4,939 ER-negative cases and 14,352 controls, combined with 7,333 ER-negative cases and 42,468 controls and 15,252 BRCA1 mutation carriers genotyped on the iCOGS array. We identify four previously unidentified loci including two loci at 13q22 near KLF5, a 2p23.2 locus near WDR43 and a 2q33 locus near PPIL3 that display genome-wide significant associations with ER-negative breast cancer. In addition, 19 known breast cancer risk loci have genome-wide significant associations and 40 had moderate associations (P<0.05) with ER-negative disease. Using functional and eQTL studies we implicate TRMT61B and WDR43 at 2p23.2 and PPIL3 at 2q33 in ER-negative breast cancer aetiology. All ER-negative loci combined account for ∼11% of familial relative risk for ER-negative disease and may contribute to improved ER-negative and BRCA1 breast cancer risk prediction

Refined histopathological predictors of BRCA1 and BRCA2 mutation status : a large-scale analysis of breast cancer characteristics from the BCAC, CIMBA, and ENIGMA consortia

Abstract Introduction The distribution of histopathological features of invasive breast tumors in BRCA1 or BRCA2 germline mutation carriers differs from that of individuals with no known mutation. Histopathological features thus have utility for mutation prediction, including statistical modeling to assess pathogenicity of BRCA1 or BRCA2 variants of uncertain clinical significance. We analyzed large pathology datasets accrued by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and the Breast Cancer Association Consortium (BCAC) to reassess histopathological predictors of BRCA1 and BRCA2 mutation status, and provide robust likelihood ratio (LR) estimates for statistical modeling. Methods Selection criteria for study/center inclusion were estrogen receptor (ER) status or grade data available for invasive breast cancer diagnosed younger than 70 years. The dataset included 4,477 BRCA1 mutation carriers, 2,565 BRCA2 mutation carriers, and 47,565 BCAC breast cancer cases. Country-stratified estimates of the likelihood of mutation status by histopathological markers were derived using a Mantel-Haenszel approach. Results ER-positive phenotype negatively predicted BRCA1 mutation status, irrespective of grade (LRs from 0.08 to 0.90). ER-negative grade 3 histopathology was more predictive of positive BRCA1 mutation status in women 50 years or older (LR = 4.13 (3.70 to 4.62)) versus younger than 50 years (LR = 3.16 (2.96 to 3.37)). For BRCA2, ER-positive grade 3 phenotype modestly predicted positive mutation status irrespective of age (LR = 1.7-fold), whereas ER-negative grade 3 features modestly predicted positive mutation status at 50 years or older (LR = 1.54 (1.27 to 1.88)). Triple-negative tumor status was highly predictive of BRCA1 mutation status for women younger than 50 years (LR = 3.73 (3.43 to 4.05)) and 50 years or older (LR = 4.41 (3.86 to 5.04)), and modestly predictive of positive BRCA2 mutation status in women 50 years or older (LR = 1.79 (1.42 to 2.24)). Conclusions These results refine likelihood-ratio estimates for predicting BRCA1 and BRCA2 mutation status by using commonly measured histopathological features. Age at diagnosis is an important variable for most analyses, and grade is more informative than ER status for BRCA2 mutation carrier prediction. The estimates will improve BRCA1 and BRCA2 variant classification and inform patient mutation testing and clinical management

Identification of four novel susceptibility loci for oestrogen receptor negative breast cancer

Common variants in 94 loci have been associated with breast cancer including 15 loci with genome-wide significant associations (PPeer reviewe

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA

Mechanical Compressibility of the Glycosylphosphatidylinositol (GPI) Anchor Backbone Governed by Independent Glycosidic Linkages

About 1% of the human proteome is anchored to the outer leaflet of cell membranes via a class of glycolipids called GPI anchors. In spite of their ubiquity, experimental information about the conformational dynamics of these glycolipids is rather limited. Here, we use a variety of computer simulation techniques to elucidate the conformational flexibility of the Man-α(1→2)-Man-α(1→6)-Man-α(1→4)-GlcNAc-α-OMe tetrasaccharide backbone <b>2</b> that is an essential and invariant part of all GPI-anchors. In addition to the complete tetrasaccharide structure, all disaccharide and trisaccharide subunits of the GPI backbone have been studied as independent moieties. The extended free energy landscape as a function of the corresponding dihedral angles has been determined for each glycosidic linkage relevant for the conformational preferences of the tetrasaccharide backbone (Man-α(1→2)-Man, Man-α(1→6)­Man and Man-α(1→4)-GlcNAc). We compared the free energy landscapes obtained for the same glycosidic linkage within different oligosaccharides. This comparison reveals that the conformational properties of a linkage are primarily determined by its two connecting carbohydrate moieties, just as in the corresponding disaccharide. Furthermore, we can show that the torsions of the different glycosidic linkages within the GPI tetrasaccharide can be considered as statistically independent degrees of freedom. Using this insight, we are able to map the atomistic description to an effective, reduced model and study the response of the tetrasaccharide <b>2</b> to external forces. Even though the backbone assumes essentially a single, extended conformation in the absence of mechanical stress, it can be easily bent by forces of physiological magnitude

COPII Coat Composition Is Actively Regulated by Luminal Cargo Maturation

SummaryBackgroundExport from the ER is an essential process driven by the COPII coat, which forms vesicles at ER exit sites (ERESs) to transport mature secretory proteins to the Golgi. Although the basic mechanism of COPII assembly is known, how COPII machinery is regulated to meet varying cellular secretory demands is unclear.ResultsHere, we report a specialized COPII system that is actively recruited by luminal cargo maturation. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are luminal secretory proteins anchored to the membrane by the glycolipid GPI. After protein attachment in the ER lumen, lipid and glycan parts of the GPI anchor are remodeled. In yeast, GPI-lipid remodeling concentrates GPI-APs into specific ERESs. We found that GPI-glycan remodeling induces subsequent recruitment of the specialized ER export machinery that enables vesicle formation from these specific ERESs. First, the transmembrane cargo receptor p24 complex binds GPI-APs as a lectin by recognizing the remodeled GPI-glycan. Binding of remodeled cargo induces the p24 complex to recruit the COPII subtype Lst1p, specifically required for GPI-AP ER export.ConclusionsOur results show that COPII coat recruitment by cargo receptors is not constitutive but instead is actively regulated by binding of mature ligands. Therefore, we reveal a novel functional link between luminal cargo maturation and COPII vesicle budding, providing a mechanism to adjust specialized COPII vesicle production to the amount and quality of their luminal cargos that are ready for ER exit. This helps to understand how the ER export machinery adapts to different needs for luminal cargo secretion