The mRNA-binding site of annexin A2 resides in helices C-D of its domain IV

Annexin A2 (AnxA2) is a Ca(2+)-binding and phospholipid-binding protein involved in different intracellular processes including exocytosis, endocytosis and membrane-cytoskeleton movements. We have previously identified AnxA2 as an mRNA-binding protein present in cytoskeleton-bound polysomes, that binds to a specific approximately 100 nucleotide region in the 3'-untranslated region of c-myc and its cognate mRNA. In the present study, we show by UV cross-linking assays and surface plasmon resonance analyses that the mRNA-binding site of AnxA2 resides in its domain IV. Furthermore, the interaction of full-length AnxA2 with the 3'-untranslated region of anxA2 mRNA is Ca(2+)-dependent. By contrast, the interaction is Ca(2+)-independent for the isolated domain IV of AnxA2, suggesting that the mRNA-binding site is masked in Apo-AnxA2 and gains exposure through a Ca(2+)-induced conformational change of AnxA2 generating a favourable mRNA-binding site. The AnxA2-mRNA interaction is specific and involves helices C and D in domain IV of AnxA2, since point mutagenesis of several charged and polar exposed residues of these helices in the full-length protein strongly reduce RNA binding. The interaction appears to be sequential involving an initial phase of recognition dominated by electrostatic interactions, most likely between lysine residues and the phosphate backbone of RNA, followed by a second phase contributing to the specificity of the interaction.     

N-linked glycan modifications in gp120 of human immunodeficiency virus type 1 subtype C render partial sensitivity to 2G12 antibody neutralization

The monoclonal antibody (MAb) 2G12 recognizes a cluster of high-mannose oligosaccharides on the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 and is one of a select group of MAbs with broad neutralizing activity. However, subtype C viruses are generally resistant to 2G12 neutralization. This has been attributed to the absence of a glycosylation site at position 295 in most subtype C gp120s, which instead is typically occupied by a Val residue. Here we show that N-linked glycans in addition to the one at position 295 are important in the formation of the 2G12 epitope in subtype C gp120. Introduction of the glycosylation site at position 295 into three subtype C molecular clones, Du151.2, COT9.6, and COT6.15, did increase 2G12 binding to all three mutagenized gp120s, but at various levels. The COT9-V295N mutant showed the strongest 2G12 binding and was the only mutant to become sensitive to 2G12 neutralization, although very high antibody concentrations were required. Introduction of a glycosylation site at position 448 into mutant COT6-V295N, which occurs naturally in COT9, resulted in a virus that was partially sensitive to 2G12. Interestingly, a glycosylation site at position 442, which is common among subtype C viruses, also contributed to the 2G12 epitope. The addition of this glycan increased virus neutralization sensitivity to 2G12, whereas its deletion conferred resistance. Collectively, our results indicate that the 2G12 binding site cannot readily be reconstituted on the envelopes of subtype C viruses, suggesting structural differences from other HIV subtypes in which the 2G12 epitope is naturally expressed.     

4-Aminothiazolyl analogs of GE2270 A: design, synthesis and evaluation of imidazole analogs

Imidazole analogs of the antibiotic natural product GE2270 A (1) were designed, synthesized, and evaluated for Gram positive bacteria growth inhibition. A recently reported, copper-mediated synthesis was exploited to prepare 4-thiazolyl imidazole analogs of 1. The synthesis described represents a structurally complex, natural product-based application of this recently reported synthetic methodology. In addition, the biological evaluation of the imidazole-based analogs further define the SAR of the 4-aminothiazolyl-based antibacterial template.     

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

Background  

The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella.     

The architecture of gene regulatory variation across multiple human tissues: the MuTHER study

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis--MCTA) permits immediate replication of eQTLs using co-twins (93%-98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%-20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.     

Mapping of signaling networks through synthetic genetic interaction analysis by RNAi

The analysis of synthetic genetic interaction networks can reveal how biological systems achieve a high level of complexity with a limited repertoire of components. Studies in yeast and bacteria have taken advantage of collections of deletion strains to construct matrices of quantitative interaction profiles and infer gene function. Yet comparable approaches in higher organisms have been difficult to implement in a robust manner. Here we report a method to identify genetic interactions in tissue culture cells through RNAi. By performing more than 70,000 pairwise perturbations of signaling factors, we identified >600 interactions affecting different quantitative phenotypes of Drosophila melanogaster cells. Computational analysis of this interaction matrix allowed us to reconstruct signaling pathways and identify a conserved regulator of Ras-MAPK signaling. Large-scale genetic interaction mapping by RNAi is a versatile, scalable approach for revealing gene function and the connectivity of cellular networks.