Allosteric regulation of γ-secretase activity by a phenylimidazole-type γ-secretase modulator.

γ-Secretase is an intramembrane-cleaving protease responsible for the generation of amyloid-ß (Aß) peptides. Recently, a series of compounds called γ-secretase modulators (GSMs) has been shown to decrease the levels of long toxic Aß species (i.e., Aß42), with a concomitant elevation of the production of shorter Aß species. In this study, we show that a phenylimidazole-type GSM allosterically induces conformational changes in the catalytic site of γ-secretase to augment the proteolytic activity. Analyses using the photoaffinity labeling technique and systematic mutational studies revealed that the phenylimidazole-type GSM targets a previously unidentified extracellular binding pocket within the N-terminal fragment of presenilin (PS). Collectively, we provide a model for the mechanism of action of the phenylimidazole-type GSM in which binding at the luminal side of PS induces a conformational change in the catalytic center of γ-secretase to modulate Aß production.

Crystal structure of the sodium-potassium pump at 2.4 A resolution.

Sodium-potassium ATPase is an ATP-powered ion pump that establishes concentration gradients for Na(+) and K(+) ions across the plasma membrane in all animal cells by pumping Na(+) from the cytoplasm and K(+) from the extracellular medium. Such gradients are used in many essential processes, notably for generating action potentials. Na(+), K(+)-ATPase is a member of the P-type ATPases, which include sarcoplasmic reticulum Ca(2+)-ATPase and gastric H(+), K(+)-ATPase, among others, and is the target of cardiac glycosides. Here we describe a crystal structure of this important ion pump, from shark rectal glands, consisting of alpha- and beta-subunits and a regulatory FXYD protein, all of which are highly homologous to human ones. The ATPase was fixed in a state analogous to E2.2K(+).P(i), in which the ATPase has a high affinity for K(+) and still binds P(i), as in the first crystal structure of pig kidney enzyme at 3.5 A resolution. Clearly visualized now at 2.4 A resolution are coordination of K(+) and associated water molecules in the transmembrane binding sites and a phosphate analogue (MgF(4)(2-)) in the phosphorylation site. The crystal structure shows that the beta-subunit has a critical role in K(+) binding (although its involvement has previously been suggested) and explains, at least partially, why the homologous Ca(2+)-ATPase counter-transports H(+) rather than K(+), despite the coordinating residues being almost identical.

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain.

The sodium-potassium pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.

Occludin-binding single-chain variable fragment and antigen-binding fragment antibodies prevent hepatitis C virus infection.

Occludin (OCLN) is a tetraspan membrane component of epithelial tight junctions and a known receptor for hepatitis C virus (HCV). Previously, we established functional monoclonal antibodies (mAbs) that bind to each extracellular loop of OCLN and showed their ability to prevent in vitro and in vivo HCV infection. In this study, we converted these mAbs to corresponding monovalent antigen-binding fragments (Fabs) and single-chain variable fragment (scFv) antibodies. These Fab fragments and scFv antibodies demonstrate similar binding specificity and affinity to parental anti-OCLN mAbs. Moreover, Fab fragments and scFv antibodies inhibit in vitro HCV infection. The small functional monovalent OCLN-binding probes reported in our study have high potential as drug candidates and tools for biological and pharmaceutical studies of OCLN.

Claudin-2 binding peptides, VPDSM and DSMKF, down-regulate claudin-2 expression and anticancer resistance in human lung adenocarcinoma A549 cells.

Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in spheroid culture models of human lung adenocarcinoma A549 cells. However, there is no chemical which can improve the sensitivity to anticancer drugs. So far, we reported that DFYSP, a short peptide which mimics the second extracellular loop (ECL2) of CLDN2, decreases CLDN2 expression in A549 cells, but the concentration is relatively high. Here, we found that the effects of VPDSM and DSMKF are stronger than that of DFYSP. Both VPDSM and DSMKF decreased the protein levels of CLDN2 without affecting the mRNA levels of CLDN2. The peptide-induced decrease in CLDN2 expression was suppressed by monodansylcadaverine (MDC), a clathrin-dependent endocytosis (CDE) inhibitor, and chloroquine, a lysosome inhibitor. CLDN2 was colocalized with ZO-1, an adapter protein, in tight junctions (TJs) under control conditions, whereas it disappeared from the TJs in the peptide-treated cells. Quartz crystal microbalance assay showed that both peptides can bind to recombinant CLDN2 protein. Both peptides increased permeability to paracellular transport marker lucifer yellow. In three-dimensional spheroid culture models, both peptides enhanced the sensitivity to doxorubicin, a cytotoxic anticancer drug, which was inhibited by MDC. We suggest that VPDSM and DSMKF enhance the chemosensitivity to anticancer drugs in aggregated adenocarcinoma cells mediated by the CDE pathway and lysosomal degradation of CLDN2 in lung adenocarcinoma cells. VPDSM and DSMKF, which mimic the ECL2 of CLDN2, may become novel adjuvant therapeutic drugs for lung adenocarcinoma.

Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin.

The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Šresolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs.

Cell-free methods to produce structurally intact mammalian membrane proteins.

The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin.

Clearance of bacterial lipopolysaccharides and lipid A by the liver and the role of argininosuccinate synthase.

The liver is thought to be involved in the systemic clearance and detoxification of lipopolysaccharide (LPS). Argininosuccinate synthase (AS), a liver cytosolic urea cycle enzyme, has been found to bind to and inactivate LPS and lipid A. To elucidate the participation of AS in the clearance of LPS by liver and hepatocytes, we investigated the correlation between AS content and the removal of lipid A and LPS in vivo and in vitro, tracing levels of biological activity. A hepatotoxic model in which mice were injected with CCl(4) revealed a significant reduction in lipid A clearance along with liver failure on day 1; total body clearance was changed to 0.534 ml/min from 1.42 ml/min. AS content in liver concomitantly decreased to about half and AS leaked to blood at about 6 microg/ml. Total body clearance of i.v. injected AS was estimated at 0.083 ml/min, which predicted about 24-h leakage of AS after CCl(4) injection. The treatment also reduced the clearance of R-type LPSs to a lesser degree the larger its polysaccharide portion. S-type LPS, which has a large O-antigen polysaccharide, exhibited enhancement of clearance on CCl(4) treatment. When pretreated in vitro with AS and injected into normal mice, lipid A and R-type LPS showed a similar pattern of clearance of residual activities to the untreated forms, but S-type LPS exhibited enhancement of clearance. Comparison between different strains of mice revealed a correlation of AS content in liver and lipid A clearance, where the higher AS strain C3H/He mice showed a more rapid clearance than the lower AS strains C57BL/6 and BALB/c. Primary spheroid cultures of hepatocytes treated with 0.1 microM dexamethasone and 1 microM glucagon showed about a 2-fold increase in AS amount and a more rapid clearance of LPS from culture medium than untreated cells. These results suggest that AS in hepatocytes may be involved in the process of lipid A and LPS clearance and the extracellular leakage of AS may also participate in the systemic detoxification.