Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2

Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signalling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. While the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC:BMP binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2—collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analysed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs.This work was supported by Cambridge Overseas Trust and China Scholarship Council through a postgraduate scholarship to E-R. X

Genome-Wide Identification of Alternative Splice Forms Down-Regulated by Nonsense-Mediated mRNA Decay in Drosophila

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly

Improving manganese circular economy from cellulose by chelation with siderophores immobilized to magnetic microbeads

Manganese (Mn) contained in cellulose is partially responsible for an increased consumption of paper bleaching chemicals (like O₂, H₂O₂), consequently diminishing the efficiency in pulp processing, darkening the pulp and deteriorating pulp quality. Usually, Mn in the paper industry is removed employing the environmentally critical EDTA. A greener alternative constitutes, however, the use of siderophores, high-affinity metal-chelating organic compounds that are produced by microorganisms to acquire metals (Fe and Mn among others), like desferrioxamine B (DFOB) or desferrioxamine E (DFOE). The use of native Mn-transporter proteins, like PratA, constitutes another possibility for Mn removal. The evaluation of utilizing siderophores or PratA for Mn removal from cellulose in a circular economy scheme is therefore essential. Firstly, Mn removal from cellulose was performed by immobilizing siderophores or PratA on magnetic beads (M-PVA C22). Secondly, the beads were incubated overnight with a 2% cellulose suspension, allowing Mn-ligand complex formation. Finally, cellulose suspensions were submitted for Mn quantification, employing either the TCPP [Tetrakis(4-carboxyphenyl)porphyrin] method, the PAN [1-(2-pyridylazo)-2-naphthol] method or the Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). When non-immobilized ligands were employed, a 31% Mn removal was achieved; when using immobilized ligands, around 10% Mn removal was obtained. Treated and untreated cellulose was analyzed by SEM and the Mn distribution between the solid and liquid phase was parameterized using adsorption isotherm models. This novel greener method proved to be feasible and easy, leading to potential improvements in the paper industry. Next research steps are to optimize Mn removal and quantify Mn recovery after ligand decoupling before scaling-up