Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6°C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.Intrinsic membrane proteins are essential for the selective transport of molecules in and out of cells and hence the regulation of their concentration within the cell. Aquaporins (AQPs) 2 primarily facilitate the transport of water across biological membranes, where water movements are driven by hypertonic or hypotonic conditions. AQPs consist of six transmembrane domains and five connecting loops ( Fig. 1) with both termini located on the intracellular side of the membrane. Aquaporins also contain two half-helices in loops B and E which align to form a pseudo-transmembrane helix, the focus of which contains the signature motif asparagine-proline-alanine (NPA). In vivo the protein assemblies into homotetramers, and each monomer functions as a water channel. Thirteen aquaporin family members exist within humans, with varying tissue distribution and substrate specificity (1). Two major subclasses have been identified: the orthodox aquaporins (hAQP0, hAQP1, hAQP2, hAQP4, hAQP5, hAQP6, and hAQP8) mainly facilitating the movement of water, and the aquaglyceroporins (hAQP3, hAQP7, hAQP9, and hAQP10) which facilitate both the transport of water and of other small solutes.Human aquaporin10 (hAQP10) was first identified in a human jejunum cDNA library and was observed to have 264 amino acids and dual copies of the aquaporin signature NPA motifs, confirming it as a member of the aquaporin family located in the small intestine (2). From homology studies the protein had been classified as an aquaglyceroporin, but surprisingly it did not show any glycerol or urea transport and only a low permeability to water in Xenopus oocytes (2). Shortly thereafter, an independent study identified...
Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 μm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6–2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).
With the public availability of biochemical assays and screening data constantly increasing, new applications for data mining and method analysis are evolving in parallel. One example is BioAssay Ontology (BAO) for systematic classification of assays based on screening setup and metadata annotations. In this article we report a high-throughput screening (HTS) against phospho-N-acetylmuramoyl-pentapeptide translocase (MraY), an attractive antibacterial drug target involved in peptidoglycan synthesis. The screen resulted in novel chemistry identification using a fluorescence resonance energy transfer assay. To address a subset of the false positive hits, a frequent hitter analysis was performed using an approach in which MraY hits were compared with hits from similar assays, previously used for HTS. The MraY assay was annotated according to BAO and three internal reference assays, using a similar assay design and detection technology, were identified. Analyzing the assays retrospectively, it was clear that both MraY and the three reference assays all showed a high false positive rate in the primary HTS assays. In the case of MraY, false positives were efficiently identified by applying a method to correct for compound interference at the hit-confirmation stage. Frequent hitter analysis based on the three reference assays with similar assay method identified additional false actives in the primary MraY assay as frequent hitters. This article demonstrates how assays annotated using BAO terms can be used to identify closely related reference assays, and that analysis based on these assays clearly can provide useful data to influence assay design, technology, and screening strategy.
This paper describes and analyses the EU Regulation establishing a framework for the screening of foreign direct investments (FDI) into the Union.The negotiations that preceded the adoption of the Regulation were characterized by greatly divergent views of Member States on the proper use of FDI screening. Under such conditions, the Regulation’s framework for administrative cooperation and information sharing was as much as the Commission could hope for. The main challenge going forward lies in balancing the policy priorities of individual Member States in determining what constitutes a threat to security and public order, with the needs of the collective, that is the EU. In that respect, the indicative criteria set out in the Regulation may initiate a drive towards a rough consensus. While any ambition to fully harmonize FDI screening has now become only a long-term goal, this paper finds that the Regulation has established a nascent EU screening mechanism, albeit a well-camouflaged one.
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