Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigour of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and nuclear magnetic resonance. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle–host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.
A systematic analysis of the dimerization, membrane remodelling and higher order assembly properties of all 12 human SNX-BAR sorting nexins reveals how different SNX-BAR combinations allow the formation of distinct tubular subdomains from the same endosomal vacuole during cargo sorting.
Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine (PE) in the double-membrane phagophore is a key event in the early phase of macroautophagy, but can also occur on single membrane structures. In both cases this involves transfer of LC3/GABARAP from ATG3 to PE at the target membrane. Here we have purified the full-length human ATG12-5-16L1 complex and show its essential role in LC3B/GABARAP lipidation in vitro. We have identified two functionally distinct membrane-binding regions in ATG16L1. An N-terminal membranebinding amphipathic helix is required for LC3B lipidation under all conditions tested. In contrast, the C-terminal membrane-binding region is dispensable for canonical autophagy, but essential for VPS34-independent LC3B-lipidation at perturbed endosomes. We further show that the ATG16L1 C-terminus can compensate for WIPI2 depletion to sustain lipidation during starvation. Remarkably, the C-terminal membrane-binding region comprises the β-isoform-specific sequence of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish LC3B-lipidation under different cellular conditions.
Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the ␣-and 2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.The family name sorting nexin (SNX) 1 is given to a large group of proteins that are represented throughout the eukaryotic kingdom. Sorting nexins are characterized by the presence of a subclass of the phosphoinositide-binding phox (PX) domain, and it is believed that a common function of proteins in this family is to participate in sorting processes in the cell. Several members localize to endosomal structures, and some of the SNX proteins have been shown to directly interact with transmembrane receptors to regulate their sorting in the endosomal pathway (for a review, see Ref. 1). The localization of SNX proteins is suggested to be determined by their PX domains, although protein-protein interactions may also contribute to the membrane specificity (2, 3).SNX9 and its close relative SNX18 are the only members of the sorting nexin family that contains an Src homology 3 (SH3) domain. SH3 domains interact specifically with short prolinecontaining sequences (PXXP) and are present in a large number of proteins (4). Target sequences are often located in distinct domains, referred to as proline-rich domains (PRDs). SNX9 was originally identified as a molecule that interacted with PRDs in certain metalloprotease disintegrins (ADAMs) (5). Work in Drosophila showed that SNX9 (named DSH3PX1) interacted with Dock (the fly orthologue of mammalian Nck) and Dscam (Down's syndrome cell adhesion molecule) to form a complex involved in axonal guidance in the fly (6, 7). In addition, SNX9 was found to interact with the clathrin-binding tyrosine kinase Ack through an SH...
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