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ABCB1, also called MDR1 or P‐glycoprotein, exports various hydrophobic compounds and plays an essential role as a protective physiological barrier in several organs, including the brain, testis, and placenta. However, little is known about the structural mechanisms that allow ABCB1 to recognize hydrophobic compounds of diverse structures or the coupling of ATP hydrolysis to uphill substrate export. High‐resolution X‐ray crystal structures of the pre‐ and post‐transport states and FRET analyses in living cells have revealed that an aromatic hydrophobic network at the top of the inner cavity is key for the conformational change in ABCB1 that is triggered by a hydrophobic substrate. ATP binding, but not hydrolysis, induces a progressive network that results in a twisting motion of the whole protein, squeezing out the substrate directly to the extracellular space. This twist‐and‐squeeze mechanism by which ABCB1 exports hydrophobic substrates is distinct from those of other transporters. [ABSTRACT FROM AUTHOR]

Recent progress in understanding the essential roles of mechanical forces in regulating various cellular processes expands the field of biology to one where interdisciplinary approaches with engineering techniques become indispensable. Contractile forces or contractility-inherently present in proliferative cells due to the activity of ubiquitous nonmuscle myosin II ( NMII)-are one of such mechano-regulators, but because NMII works downstream of diverse signaling pathways, it is often difficult to predict how the inherent cellular forces change upon perturbations to particular molecules. Here, we determine whether the contractility of individual cells is upregulated or downregulated based on an assay analyzing specific deformations of silicone gel substrates. We focus on the effect of mutations in the human MYH9 gene that encodes NMIIA, which have been implicated in the pathogenesis of various diseases including nephritis. Our assay equipped with a high-throughput data analysis capability reveals that a point mutation of E1841K but not I1816V significantly reduces the magnitude of the endogenous forces of human embryonic kidney ( HEK293) cells. Given the increasingly recognized roles of the endogenous forces as a critical mechano-regulator as well as that no apparent morphological changes were induced to cells even by introducing the mutations, our findings suggest a possibility that the detected reduction in the force magnitude at the individual cellular level may underlie the pathogenesis of the kidney disease. [ABSTRACT FROM AUTHOR]

High cholesterol turnover catalyzed by cholesterol 24-hydroxylase is essential for neural functions, especially learning. Because 24(S)-hydroxycholesterol (24- OHC), produced by 24-hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH- SY5Y neuron-like cells as a model, we examined whether 24- OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24- OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor ( LXR/ RXR). When the expression of ABCA1 and ABCG1 was induced, 24- OHC efflux was stimulated in the presence of high-density lipoprotein ( HDL), whereas apolipoprotein A-I was not an efficient acceptor. The efflux was suppressed by the addition of si RNA against ABCA1, but not by ABCG1 si RNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24- OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A-I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/ RXR ligands suppressed the toxicity of 24- OHC. These results suggest that ABCA1 actively eliminates 24- OHC in the presence of HDL as a lipid acceptor and protects neuronal cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] Dlg5 interacts with both the TGF-β type I and type II receptors. [•] Dlg5 colocalizes with TGF-β receptors at the plasma membrane. [•] Dlg5 promotes TGF-β type I receptor degradation. [•] These findings can explain how Dlg5 regulates TGF-β signaling. [ABSTRACT FROM AUTHOR]

ATP-binding cassette protein A1 (ABCA1) mediates transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL). It is currently unknown to what extent ABCA1 endocytosis and recycling contribute to the HDL formation. To address this issue, we expressed human ABCA1 constructs with either an extracellular HA tag or an intracellular GFP tag in cells, and used this system to characterize endocytosis and recycling of ABCA1 and apoA-I. Under basal conditions, ABCA1 and apoA-I are endocytosed via a clathrin- and Rab5-mediated pathway and recycled rapidly back to the cell surface, at least in part via a Rab4-mediated route; approximately 30% of the endocytosed ABCA1 is recycled back to the cell surface. When receptor-mediated endocytosis is inhibited, the level of ABCA1 at the cell surface increases and apoA-I internalization is blocked. Under these conditions, apoA-I mediated cholesterol efflux from cells that have accumulated lipoprotein-derived cholesterol is decreased, whereas efflux from cells without excess cholesterol is increased. These results suggest that the retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration and oncogenesis. In this study, we found that exogenous expression of vinexin β enhanced the phosphorylation of 180-kDa proteins in an EGF-dependent manner in Cos-7 cells. Western blot analysis using phospho-specific antibodies against EGFR identified EGFR as a phosphorylated 180-kDa protein. Vinexin β did not stimulate the phosphorylation of EGFR but suppressed the dephosphorylation, resulting in a sustained phosphorylation. Mutational analyses revealed that both the first and third SH3 domains were required for a sustained phosphorylation of EGFR. Small interfering RNA-mediated knockdown of vinexin β reduced the phosphorylation of EGFR on the cell surface in HeLa cells. The sustained phosphorylation of EGFR induced by vinexin β was completely abolished by adding the EGFR-specific inhibitor AG1478 even after EGF stimulation, suggesting that the kinase activity of EGFR is required for the sustained phosphorylation induced by vinexin β. We also found that E3 ubiquitin ligase c-Cbl is a binding partner of vinexin β through the third SH3 domain. Expression of wild-type vinexin β but not a mutant containing a mutation in the third SH3 domain decreased the cytosolic pool of c-Cbl and increased the amount of membrane-associated c-Cbl. Furthermore, over-expression of c-Cbl suppressed the sustained phosphorylation of EGFR induced by vinexin β. These results suggest that vinexin β plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl. [ABSTRACT FROM AUTHOR]

Abstract: Non-receptor tyrosine kinase Abl is a well known regulator of the actin-cytoskeleton, including the formation of stress fibers and membrane ruffles. Vinexin is an adapter protein consisting of three SH3 domains, and involved in signal transduction and the reorganization of actin cytoskeleton. In this study, we found that vinexin α as well as β interacts with c-Abl mainly through the third SH3 domain, and that vinexin and c-Abl were colocalized at membrane ruffles in rat astrocytes. This interaction was reduced by latrunculin B, suggesting an F-actin-mediated regulatory mechanism. We also found that vinexin α but not β was phosphorylated at tyrosine residue when c-Abl or v-Abl was co-expressed. A mutational analysis identified tyrosine 127 on vinexin α as a major site of phosphorylation by c- or v-Abl. These results suggest that vinexin α is a novel substrate for Abl. [Copyright &y& Elsevier]

The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin β increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin β with WAVE2 as well as WAVE1 and N-WASP. Vinexin β interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin β. Mutations disrupting the interaction impaired the ability of vinexin β to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin β. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin β. These results suggest that vinexin β regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner. [ABSTRACT FROM AUTHOR]

The src homology 2 (SH2) domain-containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5) P3] to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4) P2]. We report the identification of the cytoskeletal protein Vinexin as a protein interacting with SHIP2. This was achieved by yeast two-hybrid screening using the C-terminal region of SHIP2 as bait. Vinexin has previously been identified as a vinculin-binding protein that plays a key role in cell spreading and cytoskeletal organization. The interaction between SHIP2 and Vinexin was confirmed in lysates of both COS-7 cells and mouse embryonic fibroblasts (MEF). The C-terminus was involved in the interaction, as shown by the transfection of a truncated C-terminus mutant of SHIP2. In addition, we showed the colocalization between Vinexin α and SHIP2 at the periphery of transfected COS-7 cells. When added in vitro to SHIP2, Vinexin did not affect the PtdIns(3,4,5) P3 5-phosphatase activity of SHIP2. Enhanced cell adhesion to collagen-I-coated dishes was shown upon transfection of either SHIP2 or Vinexin to COS-7 cells. This effect was no longer observed with either a catalytic mutant or the C-terminus mutant of SHIP2. It also appears SHIP2 specific; this was not seen with SHIP1. Adhesion to the same matrix was decreased in SHIP2–/– MEF cells compared with MEF+/+ cells. Our data suggest that SHIP2 interaction with Vinexin promotes the localization of SHIP2 at the periphery of the cells leaving its catalytic site intact. The complex formation between Vinexin and SHIP2 may increase cellular adhesion. The data reinforce the concept that SHIP2 is active both as a PtdIns(3,4,5) P3 5-phosphatase and as a modulator of focal contact formation. [ABSTRACT FROM AUTHOR]

ABCA7 is a member of the subfamily A of adenosine triphosphate-binding cassette (ABC) proteins, and highly homologous to ABCA1, which mediates the release of cellular cholesterol and phospholipid to form high-density lipoprotein. ABCA1 and ABCA7 contain two large extracellular domains, ECD1 and 2, which are thought to be important for their functions. Interestingly, part of ECD1 of ABCA7 is deposited as an autoantigen of Sjögren's syndrome. To determine the relationship between ABCA7 and Sjögren's syndrome, an immunohistochemical study was conducted with salivary gland biopsy samples from patients with Sjögren's syndrome. ECD1 of human ABCA7 (amino acids 45–549) was expressed in Escherichia coli as a protein fused with glutathione-S-transferase and a monoclonal antibody, KM3095, was generated. KM3095-immunoreactive cells were observed in salivary glands from 10 of 18 patients with Sjögren's syndrome. Immunostaining of serial sections with the plasma cell marker NCL-PC indicated that most of the plasma cells infiltrating salivary glands of patients with Sjögren's syndrome were KM3095-immunoreactive. Although the pathological or biological significance is not clear, it will be intriguing to further examine the relationship between ABCA7 and Sjögren's syndrome. [ABSTRACT FROM AUTHOR]