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A central question in Wnt signaling is the regulation of β-catenin phosphorylation and degradation. Multiple kinases, including CKIα and GSK3, are involved in β-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of β-catenin. However, which phosphatase dephosphorylates β-catenin in vivo and how the specificity of β-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates β-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/β-catenin signaling in Drosophila. Moreover, we have identified PR55α as the regulatory subunit of PP2A that controls β-catenin phosphorylation and degradation. PR55α, but not the catalytic subunit, PP2Ac, directly interacts with β-catenin. RNA interference knockdown of PR55α elevates β-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55α enhances Wnt signaling. Taken together, our results suggest that PR55α specifically regulates PP2A-mediated β-catenin dephosphorylation and plays an essential role in Wnt signaling.Wnt/β-catenin signaling plays essential roles in development and tumorigenesis (13). Our previous work found that β-catenin is sequentially phosphorylated by CKIα4 and GSK3 (4), which creates a binding site for β-Trcp (5), leading to degradation via the ubiquitination/proteasome machinery (3). Mutations in β-catenin or APC genes that prevent β-catenin phosphorylation or ubiquitination/degradation lead ultimately to cancer (1, 2).In addition to the involvement of kinases, protein phosphatases, such as PP1, PP2A, and PP2C, are also implicated in Wnt/β-catenin regulation. PP2C and PP1 may regulate dephosphorylation of Axin and play positive roles in Wnt signaling (6, 7). PP2A is a multisubunit enzyme (810); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (1121). Toward the goal of understanding the mechanism of β-catenin phosphorylation, we carried out siRNA screening targeting several major phosphatases, in which we found that PP2A dephosphorylates β-catenin. This is consistent with a recent study where PP2A is shown to dephosphorylate β-catenin in a cell-free system (18).PP2A consists of a catalytic subunit (PP2Ac), a structure subunit (PR65/A), and variable regulatory B subunits (PR/B, PR/B′, PR/B″, or PR/B‴). The substrate specificity of PP2A is thought to be determined by its B subunit (9). By siRNA screening, we further identified that PR55α, a regulatory subunit of PP2A, specifically regulates β-catenin phosphorylation and degradation. Mechanistically, we found that PR55α directly interacts with β-catenin and regulates PP2A-mediated β-catenin dephosphorylation in Wnt signaling.  相似文献   

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The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABAARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABAAR (δL2), but not the γGABAAR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABAARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABAA γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABAARs. The δL2 mutations did not affect GlyR or GABAAR sensitivity, respectively, to Zn2+ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (35). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)2 have received substantial attention as putative sites of ethanol action that cause its behavioral effects (612). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine3, γ-aminobutyric acid type A (GABAA), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 1520) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (2325).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (712, 18, 19, 2630). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABAARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 3440).GlyRs and GABAARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABAARs (e.g. α1β2γ2), the most predominantly expressed GABAARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABAARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (4451). Sequence alignment of α1GlyR, γGABAAR, and δGABAAR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABAAR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABAARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABAARs with corresponding non-conserved residues from the δ subunit of GABAAR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABAAR subunit (δL2), but not the γGABAAR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABAARs with δL2 also markedly increased ethanol sensitivity of GABAARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).  相似文献   

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Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)2 are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (15). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (810). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (1418) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (2730). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.  相似文献   

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Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The Km, kcat, and kcat/Km values for β-carotene as substrate were 37 μm, 3.6 min−1, and 97 mm−1 min−1, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (35). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (2328). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.  相似文献   

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