Loss of α-catenin elicits a cholestatic response and impairs liver regeneration.

The liver is unique in its capacity to regenerate after injury, during which hepatocytes actively divide and establish cell-cell contacts through cell adhesion complexes. Here, we demonstrate that the loss of α-catenin, a well-established adhesion component, dramatically disrupts liver regeneration. Using a partial hepatectomy model, we show that regenerated livers from α-catenin knockdown mice are grossly larger than control regenerated livers, with an increase in cell size and proliferation. This increased proliferation correlated with increased YAP activation, implicating α-catenin in the Hippo/YAP pathway. Additionally, α-catenin knockdown mice exhibited a phenotype reminiscent of clinical cholestasis, with drastically altered bile canaliculi, elevated levels of bile components and signs of jaundice and inflammation. The disrupted regenerative capacity is a result of actin cytoskeletal disorganisation, leading to a loss of apical microvilli, dilated lumens in the bile canaliculi, and leaky tight junctions. This study illuminates a novel, essential role for α-catenin in liver regeneration.

Structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase.

The in vitro evolution and engineering of quorum-quenching lactonases with enhanced reactivities was achieved using a thermostable GKL enzyme as a template, yielding the E101G/R230C GKL mutant with increased catalytic activity and a broadened substrate range [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C., and Yew, W. S. (2010) J. Biol. Chem. 285, 40911-40920]. This enzyme possesses the (ß/α)8-barrel fold and is a member of the PLL (phosphotriesterase-like lactonase) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acyl-homoserine lactones, which mediate the quorum-sensing pathways of bacteria. The structure of the evolved N-butyryl-l-homoserine lactone (substrate)-bound E101G/R230C GKL enzyme was determined, in the presence of the inactivating D266N mutation, to a resolution of 2.2 Å to provide an explanation for the observed rate enhancements. In addition, the substrate-bound structure of the catalytically inactive E101N/D266N mutant of the manganese-reconstituted enzyme was determined to a resolution of 2.1 Å and the structure of the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 Å, and the structures of ligand-free zinc-reconstituted wild-type, E101N, R230D, and E101G/R230C mutants of GKL were determined to resolutions of 2.1, 2.1, 1.9, and 2.0 Å, respectively. In particular, the structure of the evolved E101G/R230C mutant of GKL provides evidence of a catalytically productive active site architecture that contributes to the observed enhancement of catalysis. At high concentrations, wild-type and mutant GKL enzymes are differentially colored, with absorbance maxima in the range of 512-553 nm. The structures of the wild-type and mutant GKL provide a tractable link between the origins of the coloration and the charge-transfer complex between the α-cation and Tyr99 within the enzyme active site. Taken together, this study provides evidence of the modulability of enzymatic catalysis through subtle changes in enzyme active site architecture.

Interactions of intermediate semiquinone with surrounding protein residues at the Q(H) site of wild-type and D75H mutant cytochrome bo3 from Escherichia coli.

Selective (15)N isotope labeling of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from residues R71, H98, and Q101 and peptide nitrogens from residues R71 and H98 around the semiquinone (SQ) at the high-affinity Q(H) site. The two-dimensional ESEEM (HYSCORE) data have directly identified N(ε) of R71 as an H-bond donor carrying the largest amount of unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the Q(H) site and the strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that N(ε) of a histidine residue, presumably H75, carries most of the unpaired spin density instead of N(ε) of R71, as in wild-type bo(3). However, the detailed characterization of the weakly coupled (15)N atoms from selective labeling of R71 and Q101 in D75H was precluded by overlap of the (15)N lines with the much stronger ~1.6 MHz line from the quadrupole triplet of the strongly coupled (14)N(ε) atom of H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied to probe the contribution of R71 and Q101 to the (15)N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory-based calculations to model the available information about (1)H, (15)N, and (13)C hyperfine couplings for the Q(H) site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone's electron transfer function.

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme-copper site in cytochrome bo3, E. coli.

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme-copper dinuclear site in Escherichia coli cytochrome bo(3), a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T(1), of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T (2), of the dinuclear coupled pair Fe(III)-Cu(B)(II) in the oxidized form of QO that is too short to measure directly. In the case of the F' state, the relaxation properties of the heme-copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu(B)(II) coupled by approximately 1.5-3 cm(-1) to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine-histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli.

Cytochrome bo(3) is the major respiratory oxidase located in the cytoplasmic membrane of Escherichia coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (Q(H)). The quinone bound at the Q(H) site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the Q(H) site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1-10, does not displace ubiquinone-8 from the Q(H) site. The data clearly support the existence of a second binding site for ubiquinone, the Q(L) site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1-10 may compete for binding with quinol at the Q(L) site while, at the same time, preventing formation of the ubisemiquinone at the Q(H) site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.

A multigene family of Heteractis magnificalysins (HMgs).

Sea anemones are passive predators. They use their specialized stinging cells (nematocysts) to immobilize any prey that blunders into them. A cnida fires, everting a tubule which delivers toxins that may stick to a prey. These toxins include neurotoxins, cytotoxins, cardiotoxins and haemolysins. Heteractis magnificalysins (HMgs) belong to a family of cytolysins from the sea anemone Heteractis magnifica. HMgs are 19.5kDa basic proteins of 177 amino acids with pI values ranging from 8 to 10. From 52 cloned HMg gene sequences, we showed that HMgs are encoded by a multigene family whose members are highly homologous to each other. HMg genes are intronless, and may have arisen by gene duplication, gene conversion or mutation. By modifying the extraction procedure, we purified more natural HMg proteins from H. magnifica, thus demonstrating that H. magnifica are naturally competent to produce a large number of HMg cytolysins. Native and recombinant HMg proteins differed from each other in their amino acid sequences and biological activities. In each H. magnifica, many cytolysin isoforms are produced. H. magnifica appeared to have evolved a survival mechanism whereby a large number of cytolysins of different biological properties are produced for defense and offence.

Characterization of mutants that change the hydrogen bonding of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli.

The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function.

An EPR spin label study of the quinol oxidase, E. coli cytochrome bo3: a search for redox induced conformational changes.

A search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome bo3, E. coli, has been carried out using site directed nitroxide spin labeling (SDSL) of cysteine residues. These were positioned at R134 and R309, on loops that link helices II and III and VI and VII at the entrances to the D and K proton channels, respectively. The motional characteristics of both labels have been determined using X- and W-band EPR spectroscopy at room temperature in selected redox levels in the reaction sequence of QO with oxygen, namely, the mixed valence carbon monoxide form (COMV), the oxidized (O) and super-oxidized (PM) states. The O to PM step is accompanied by the uptake of protons through the K pathway. We find no evidence for changes in the motional characteristics of either label that are expected to be associated with helical motions at the entrances to the channels. Because kinetic studies of mutants show that the redox gating of protons occurs deep within the D channel close to the heme-copper site, the present study implies that no motion is transmitted to the ends of the helices.

Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase.

Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo(3) oxidase. The optimized protocol for expression and purification yields approximately 5 mg of the enzymatically active, uniformly (13)C,(15)N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The (13)C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo(3) oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly (13)C,(15)N-labeled membrane protein.

Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli.

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2O by 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.

The signalling pathway for BCG-induced interleukin-6 production in human bladder cancer cells.

Intravesical bacillus Calmette-Guerin (BCG) is currently the therapy of choice for superficial bladder cancer with a 60-70% response rate. Induction of cytokine production (e.g. IL-6, etc.) by BCG has been found in patient's urine in vivo as well as bladder cancer cell lines. However, the signalling mechanisms are still unclear. In this study, we investigated the effect of BCG on cAMP production and its role in regulating interleukin-6 expression in the human bladder cancer cell line, MGH. After 1 hr exposure to BCG, IL-6 gene expression in MGH cells increased by 2.5-3-fold and cAMP production increased by 8-10-fold in a time- and dose-dependent manner. BCG-induced cAMP production was inhibited by both antifibronectin antibody and an adenylate cyclase inhibitor, SQ22536 in a dose-dependent way. In the presence of SQ22536, IL-6 expression in MGH cells was also greatly reduced. Furthermore, cAMP-dependent kinase inhibitors H7 and HA1004 also inhibited BCG-induced IL-6 expression in MGH, with HA1004 being much less effective than H7. Thus, BCG induces cAMP production and may regulate interleukin-6 expression partially via a cAMP-dependent pathway in human bladder cancer cells.