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The most common mutation in cystic fibrosis (CF) is ΔF508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked ΔF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum- and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of ΔF508-CFTR. The half-life of ΔF508-CFTR is also dramatically enhanced. Dexamethasone-activated ΔF508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type- and ΔF508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects ΔF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated ΔF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.Cystic fibrosis (CF)2 is the most common life-limiting genetic disease in the United States and is due to mutations in the CFTR gene. The most common mutation, ΔF508-CFTR, results in a failure of the mutant protein to traffic properly to the apical plasma membrane of epithelial cells in the lung and other organs (1, 2). By a still unknown mechanism, the loss of correctly trafficked ΔF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane (3–5). In the CF lung, such high levels of ENaC activity are believed to cause dehydration of the airway, and the consequent proinflammatory condition that characterizes CF lung pathophysiology. Similar proinflammatory pathophysiology has been reported to characterize the lung of transgenic mice which overexpress β-ENaC (6). Operationally, it seems that when membrane-localized CFTR decreases in CF, ENaC activity at the plasma membrane increases; CF-related morbidity and mortality follow.In the case of ENaC trafficking, the process is known to be regulated by a glucocorticoid receptor/SGK1 signaling pathway affecting phosphorylation of the ubiquitin ligase E3 protein Nedd4-2 (7, 8). Fig. 1 illustrates how surface expression of ENaC is controlled by the serum- and glucocorticoid-inducible kinase SGK1, the upstream signal, and the ubiquitin E3 ligase Nedd4-2, the downstream signal. Under default conditions, Nedd4-2 suppresses ENaC surface expression by binding to ENaC via the interaction between the PPXY motifs of ENaC and WW domains on Nedd4-2. Nedd4-2 then catalyzes the ubiquitination of bound ENaC. This step targets ENaC for proteasomal degradation (9, 10). However, when Nedd4-2 is phosphorylated by SGK1, the default interaction between Nedd4-2 and ENaC is reduced, and ENaC is maintained at the plasma membrane (7, 8). The requirement for Nedd4-2 for destruction of ENaC is supported by the recent observation that siRNA against Nedd4-2 is sufficient to permit ENaC to be expressed at the plasma membrane (10). Importantly, both glucocorticoid receptor (GR) and phosphoinositide-3-kinase (PI 3-kinase) signaling pathways must be present for high levels of Na+ transport to occur. For example, treatment with the GR antagonist RU38486 (11–13) or the PI 3-kinase inhibitor LY294002 (14–16) results in a complete loss of glucocorticoid-activated ENaC activity.Open in a separate windowFIGURE 1.Schematic diagram of regulation of ENaC and CFTR by SGK1/Nedd4-2. The surface expression of ENaC is controlled by the serum/glucocorticoid inducible kinase SGK1, the upstream signal, and the neural precursor cell-expressed developmentally down-regulated isoform 2 (Nedd4-2), the downstream signal. The solid black arrows trace the signal to a point where phospho-Nedd4-2 releases ENaC, thereby saving it from default ubiquitination and proteasomal destruction. ENaC is then maintained at the plasma membrane. Glucocorticoid-activated ENaC membrane trafficking is blocked by the glucocorticoid receptor antagonist RU38486 and the PI 3-kinase inhibitor LY294002. Alternatively, silencing of endogenous Nedd4-2 by siRNA enhances ENaC trafficking to the plasma membrane. (+) indicates positive regulation, and (−) indicates negative regulation.The placement of the parenthetical (CFTR) in the SGK1/Nedd4-2 signaling pathway (Fig. 1) serves to underscore our hypothesis that CFTR itself could play an interactive or parallel role in the SGK1/Nedd4-2/ENaC-trafficking mechanism. This hypothesis seems reasonable because the regulatory effects of SGK1 and Nedd4-2 are not limited to trafficking of ENaC but also regulate several other epithelial channels and transporters (17, 18). Additionally, co-expression studies in Xenopus oocytes (19, 20) have shown that SGK1 appears to greatly enhance the functional activity of CFTR.In this report we have shown that activation of the SGK1 signaling pathway by the glucocorticoid dexamethasone results in the rescue of ΔF508-CFTR. The half-life of ΔF508-CFTR, once it reaches the plasma membrane, is also dramatically enhanced. Consistently, glucocorticoid-activated ΔF508-CFTR rescue is blocked by the GR antagonist RU38486 and by the PI 3-kinase inhibitor LY294002 as well as by knockdown of endogenous SGK1 by siRNA. We have further shown that at the downstream end of the SGK1/Nedd4-2 signaling pathway, knockdown of Nedd4-2 by siRNA also results in ΔF508-CFTR rescue. Finally, co-immunoprecipitation studies indicated that Nedd4-2 binds to both WT- and ΔF508-CFTR and that treatment with either glucocorticoid or Nedd4-2 siRNA reduces formation of Nedd4-2·CFTR complexes as well as ubiquitination of ΔF508-CFTR. Consistently, chloride transport is well correlated with the level of plasma membrane expression of ΔF508-CFTR protein. These data suggest that the glucocorticoid receptor-dependent SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells. We interpret these results to indicate that drugs affecting the SGK1/Nedd4-2 signaling pathway may be promising targets for cystic fibrosis therapeutic development.  相似文献   

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We characterized three d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; d-Gal-β1→3-d-GalNAc) and lacto-N-biose I (LNB; d-Gal-β1→3-d-GlcNAc), thus indicating that they are d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylases, subclassified as GNB/LNB phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with l-rhamnose as the acceptor in the reverse reaction using α-d-galactose 1-phosphate as the donor. The reaction product was d-galactosyl-β1→4-l-rhamnose. The enzyme also showed activity on l-mannose, l-lyxose, d-glucose, 2-deoxy-d-glucose, and d-galactose in this order. When d-glucose derivatives were used as acceptors, reaction products were β-1,3-galactosides. Kinetic parameters of phosphorolytic activity on d-galactosyl-β1→4-l-rhamnose were kcat = 45 s−1 and Km = 7.9 mm, thus indicating that these values are common among other phosphorylases. We propose d-galactosyl-β1→4-l-rhamnose phosphorylase as the name for Cphy1920 protein.Phosphorylases are a group of enzymes involved in formation and cleavage of glycoside linkage together with glycoside hydrolases and glycosyl-nucleotide glycosyltransferases (synthases). Phosphorylases, which reversibly phosphorolyze oligosaccharides to produce monosaccharide 1-phosphate, are generally intracellular enzymes showing strict substrate specificity. Physiologically, such strict substrate specificity is considered to be closely related to the environment containing bacteria possessing them. For example, d-galactosyl-β1→3-N-acetyl-d-hexosamine phosphorylase (GalHexNAcP2; EC 2.4.1.211) from Bifidobacterium longum, an intestinal bacterium, forms part of the pathway metabolizing galacto-N-biose (GNB; d-Gal-β1→3-d-GalNAc) from mucin and lacto-N-biose I (LNB; d-Gal-β1→3-d-GlcNAc) from human milk oligosaccharides, both of which are present in the intestinal environment, with GNB- and LNB-releasing enzymes and GNB/LNB transporter (1–8). Another example is cellobiose phosphorylase from Cellvibrio gilvus, which is a cellulolytic bacterium. Cellobiose phosphorylase forms an important cellulose metabolic pathway with an extracellular cellulase system producing cellobiose (9, 10).The reversible catalytic reaction of phosphorylases is one of the most remarkable features that make them suitable catalysts for practical syntheses of oligosaccharides. An oligosaccharide can be produced from inexpensive material by combining reactions of two phosphorylases, one for phosphorolyzing the material and the other for synthesizing the oligosaccharide, in one pot. Based on this idea, LNB is synthesized on a large (kg) scale using sucrose phosphorylase and GalHexNAcP (11). Practical synthesis methods of trehalose and cellobiose have also been developed (12, 13). However, only 14 kinds of substrate specificities have been reported among phosphorylases (13), thus restricting their use. Therefore, it would be useful to find a phosphorylase with novel activity.GalHexNAcP phosphorolyzes GNB and LNB to produce α-d-galactose 1-phosphate (Gal 1-P) and the corresponding N-acetyl-d-hexosamine. To date, GalHexNAcP is the only phosphorylase known to act on β-galactoside. This enzyme was first found in the cell-free extract of Bifidobacterium bifidum (14) and then in B. longum (1, 15), Clostridium perfringens (16), Propionibacterium acnes (17), and Vibrio vulnificus (18). These studies revealed that GalHexNAcPs were classified into three subgroups based on substrate preference between GNB and LNB. These subgroups are as follows: 1) galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP), showing similar activity on both GNB and LNB (B. longum and B. bifidum); 2) galacto-N-biose phosphorylase (GNBP), preferring GNB to LNB (C. perfringens and P. acnes); and 3) lacto-N-biose I phosphorylase (LNBP), preferring LNB to GNB (V. vulnificus) (18). The ternary structure of GLNBP from B. longum (GLNBPBl) has been revealed recently (19). Based on the similarity in ternary structures between GLNBPBl and β-galactosidase from Thermus thermophilus, which belongs to glycoside hydrolase family 42 (19, 20), GalHexNAcP homologs are classified as GH112 (glycoside hydrolase family 112), although phosphorylases are glycosyltransferases (21, 22).Clostridium phytofermentans is an anaerobic cellulolytic bacterium. It is found in soil and grows optimally at 37 °C (23). Its whole genome sequence has been revealed (GenBankTM accession number CP000885). The bacterium possesses three GalHexNAcP homologous genes (cphy0577, cphy1920, and cphy3030 genes; GenBankTM accession numbers are ABX40964.1, ABX42289.1, and ABX43387.1, respectively). C. phytofermentans has the ability to utilize a wide range of plant polysaccharides (23), and substrate specificities of these three gene products (Cphy0577, Cphy1920, and Cphy3030 proteins) are considered to be responsible for this ability. Furthermore, the three proteins have not been clearly categorized as GLNBP, GNBP, or LNBP, based on the phylogenetic tree shown in Fig. 1.Open in a separate windowFIGURE 1.Phylogenetic tree of GalHexNAcP homologs in GH112. Multiple alignment was performed using ClustalW2 (available on the World Wide Web). A phylogenetic tree was constructed using Treeview version 1.6.6. The proteins characterized in this study are represented with boldface letters in boxes with a heavy outline. The other proteins are numbered serially in boxes. Characterized GLNBP, GNBP, and LNBP are represented with boldface black letters on a gray background, boldface white letters on a gray background, and boldface white letters on a black background, respectively. Organisms and GenBankTM accession numbers of numbered proteins are as follows: 1, CPF0553 (C. perfringens ATCC13124, ABG83511.1) (16); 2, CPE0573 (C. perfringens str.13, BAB80279.1); 3, CPR0537 (C. perfringens SM101, ABG86710.1); 4, LnpA2 (B. bifidum JCM1254, BAE95374.1) (14, 15); 5, LnpA1 (B. bifidum JCM1254, BAD80752.1) (14, 15); 6, GLNBPBl (B. longum subsp. longum JCM 1217, BAD80751.1) (1, 16); 7, Blon_2174 (B. longum subsp. infantis ATCC 15697, ACJ53235.1); 8, BL1641 (B. longum NCC2705, AAN25428.1); 9, BLD_1765 (B. longum DJO10A, ACD99210.1); 10, GnpA (P. acnes JCM6473, AB468065) (17); 11, GnpA (P. acnes JCM6425, AB468066) (17); 12, PPA0083 (P. acnes KPA171202, AAT81843.1); 13, VV2_1091 (V. vulnificus CMCP6, AAO07997.1) (18); 14, VVA1614 (V. vulnificus YJ016, BAC97640.1); 15, Oter_1377 (Opitutus terrae PB90-1, ACB74662.1); 16, BCQ_1989 (B. cereus Q1, ACM12417.1); 17, BCAH187_A2105 (Bacillus cereus AH187, ACJ78918.1).In this study, we characterized the three proteins. We reported that two of them were GalHexNAcPs and that the other was a β-galactoside phosphorylase showing unique substrate specificity.  相似文献   

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The Δ4-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5α- or 5β-reductase. Finasteride is a mechanism-based inactivator of 5α-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-de pend ent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5β-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1·NADP+·finasteride complex determined at 1.7 Å resolution shows that it is not possible for NADPH to reduce the Δ1-2-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.The Δ4-3-ketosteroid functionality is present in many important steroid hormones, e.g. testosterone, cortisone, and progesterone. An initial step in steroid hormone metabolism is the reduction of the Δ4-ene, which in humans is mediated by steroid 5α-reductases (SRD5A1, SRD5A2) or steroid 5β-reductase (AKR1D1)3 to yield the corresponding 5α- or 5β-dihydrosteroids, respectively (1, 2). The products of these reactions are not always inactive. 5α-Reductase is responsible for the conversion of testosterone to 5α-dihydrotestosterone (5α-DHT), which is the most potent natural ligand for the androgen receptor. By contrast, in addition to being involved in bile acid biosynthesis, 5β-reductase is responsible for generating 5β-pregnanes, which are natural ligands for the pregnane-X receptor (PXR) in the liver (3, 4). PXR is involved in the induction of CYP3A4, which is responsible for the metabolism of a large proportion of drugs (5, 6). Thus both 5α-reductase and 5β-reductase are involved in the formation of potent ligands for nuclear receptors.Finasteride is a selective 5α-reductase type 2 inhibitor that reduces plasma 5α-dihydrotestosterone levels and shrinks the size of the prostate (7). It is a widely used therapeutic agent in the treatment of benign prostatic hyperplasia (8, 9), it is used in androgen deprivation therapy to treat prostate cancer (10), and it has been examined as a chemopreventive agent for hormone-dependent prostate cancer (11). Finasteride was originally thought to act as a competitive inhibitor with nanomolar affinity for 5α-reductase type 2 (12). More recently, it was found that finasteride acts as a mechanism-based inactivator of this enzyme (13). Subsequent to inhibitor binding, there is hydride transfer from the NADPH cofactor to the Δ1-2-ene double bond of finasteride. The intermediate enolate tautomerizes at the enzyme active site to form a bisubstrate analogue in which dihydrofinasteride is covalently bound to NADP+ (13). The bisubstrate analogue has subnanomolar affinity for 5α-reductase type 2 (Fig. 1). No structural information exists for 5α-reductase type 1 or type 2; therefore, it is not possible to determine how finasteride would bind to the active site of a human steroid double bond reductase in the absence of an experimentally determined crystal structure.Open in a separate windowFIGURE 1.Mechanism-based inactivation of 5α-reductase type 2 by finasteride. Adapted from Bull et al. (13). R = −C(=O)-NH2; PADPR = 2′-phosphoadenosine-5″-diphosphoribose.Human steroid 5β-reductase is a member of the aldo-keto reductase (AKR) superfamily and is formally designated (AKR1D1) (14). The AKRs are soluble NADP(H)-dependent oxidoreductases with monomeric molecular masses of 37 kDa. These enzymes are amenable to x-ray crystallography, and during the last year, we and others have reported crystal structures of ternary complexes of AKR1D1 (15–17). The ternary complexes containing steroid substrates include: AKR1D1·NADP+·testosterone (PDB: 3BUR), AKR1D1·NADP+·progesterone (PDB: 3COT), AKR1D1·NADP+·cortisone (PDB: 3CMF), and AKR1D1·NADP+·Δ4-androstene-3,17-dione (PDB: 3CAS) (17). In addition, ternary complexes containing the products 5β-dihydroprogesterone (PDB: 3CAV) and 5β-dihydrotestosterone (PDB: 3DOP) have also been described (16, 18).As part of an ongoing inhibitor screen of AKR1D1, we now report that finasteride acts as a competitive inhibitor with low micromolar affinity. Additionally, we report the x-ray crystal structure of the AKR1D1·NADP+·finasteride complex.  相似文献   

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The catalytic α-subunits of Na,K- and H,K-ATPase require an accessory β-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the β-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated β-variants were expressed in Xenopus oocytes, together with the S806C α-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E1P/E2P states. Whereas the E1P/E2P conformational equilibrium was unaffected for the shorter N-terminal deletions βΔ4 and βΔ8, we observed significant shifts toward E1P for the two larger deletions βΔ13 and βΔ29. Moreover, the reduced ΔF/F ratios of βΔ13 and βΔ29 indicated an increased reverse reaction via E2P → E1P + ADP → E1 + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E2P-specific inhibitor SCH28080, which becomes especially apparent at high concentrations (100 μm). Despite unaltered apparent Rb+ affinities, the maximal Rb+ uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the β-N terminus and α-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase β-subunit may stabilize the pump in the E2P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the β-N terminus of the closely related Na,K-ATPase does not affect the E1P/E2P distribution or pump activity, indicating that the E2P-stabilizing effect by the β-N terminus is apparently a unique property of the H,K-ATPase.The gastric H,K-ATPase fulfills the remarkable task of pumping protons against a more than 106-fold concentration gradient. H+ extrusion is coupled to countertransport of an equal number of K+ ions for each ATP molecule hydrolyzed, resulting in an electroneutral overall process (1). Characteristic for all P-type ATPases, the enzyme cycles between the two principal conformational states (E1 and E2) and the corresponding phosphointermediates (E1P and E2P), which are formed by reversible phosphorylation of an aspartate residue in the highly conserved DKTGTLT motif. According to a Post-Albers-like reaction scheme (see Fig. 1A), the conformational E1P → E2P transition converts the high H+/low K+ affinity of the cation binding pocket into a low H+/high K+ affinity binding site, hence enabling proton release into the stomach lumen and subsequent binding of extracellular K+. Because the pump faces a lumenal proton concentration of ∼150 mm (2), proton release is probably the energetically most demanding step in the reaction cycle. Thus, during the conformational E1P → E2P transition, enormous pKa changes of the H+-coordinating residues have to occur that most likely involve the rearrangement of a positively charged lysine side chain (Lys-791 in rat H,K-ATPase) (3).Open in a separate windowFIGURE 1.Post-Albers scheme (A) and cryo-EM structural representation of pig gastric H,K-ATPase in the fluoroaluminate-bound pseudo-E2P state (B). A, Post-Albers scheme of the proposed reaction cycle of the gastric H,K-ATPase. E1P/E2P conformational states giving rise to voltage jump-induced fluorescence changes of TMRM-labeled H,K-ATPase molecules are highlighted (gray box). B, structural representation based on the cryo-EM structure of the pig gastric H,K-ATPase (surface or mesh, contoured at 1 σ; EM Data Bank code 5104) and the corresponding homology model (schematic; Protein Data Bank code 3IXZ). Inset, a close-up view (from the right side of the molecule) showing the putative interaction sites of the β-subunit N terminus with the P-domain (red arrow) and αTM3 (black arrow), respectively. Color coding is indicated in the figure.All P2-type ATPases share a common catalytic α-subunit, composed of 10 transmembrane domains harboring the ion-binding sites and a large cytoplasmic loop with the nucleotide-binding domain, the phosphorylation domain (P-domain),2 and the actuator domain (A-domain) (4). However, a unique feature of K+-transporting Na,K- and H,K-ATPase enzymes is the requirement for an accessory β-subunit, which is indispensable for proper folding, maturation, and plasma membrane delivery (5, 6). Despite only 20–30% overall sequence identity between the H,K-ATPase β-subunit and the Na,K β-isoforms, the topogenic structure is similar: a short N-terminal cytoplasmic tail, followed by a single transmembrane segment and a large extracellular C-terminal domain with glycosylation sites and disulfide-bridging cysteines. Numerous studies have demonstrated that the β-subunit of the Na,K-ATPase is more than just a chaperone for the α-subunit, being also required for proper ion transport activity of the holoenzyme. In fact, it has been discovered that different cell- and tissue-specific β-isoforms have distinct effects on the cation affinities (7–9). Furthermore, it was shown that mutational changes in all three topogenic domains of the Na,K-ATPase β-subunit (10–19) as well as chemical interference with disulfide-forming cysteines in the Na,K-ATPase β-subunit ectodomain (20–22) affect the cation transport properties of the sodium pump. Finally, conformational changes in the β-subunit during the Na,K-ATPase reaction cycle were demonstrated by proteolytic digestion studies (23) and voltage clamp fluorometry (24).Far less is known about the functional significance of the single H,K-ATPase β-isoform, especially about its potential impact on cation transport (reviewed in Refs. 25 and 26). We have proven recently that E2P state-specific transmembrane interactions between residues in αTM7 and two highly conserved tyrosines in the βTM of both Na,K- and H,K-ATPase significantly stabilize the E2P conformation (19). Mutational disruptions of this interaction resulted in substantial shifts toward E1P and severely affected H+ secretion, which highlighted the physiological relevance of this E2P state stabilization. Notably, according to the recently published cryo-EM structure of pig gastric H,K-ATPase in the pseudo-E2P state (27), the N-terminal tail of the β-subunit makes direct contact with the phosphorylation domain of the α-subunit (see Fig. 1B), thus indicating an additional E2P state stabilization mediated by the β-N terminus. Although this idea was further supported by biochemical studies on N-terminally truncated mutants, direct evidence for this putative E2P-stabilizing interaction and its potential significance for ion transport in intact cells is still lacking.Here, we demonstrate for the first time the functional importance of the gastric H,K-ATPase β-subunit N terminus in living cells under in vivo conditions: voltage clamp fluorometry, Rb+ flux, and SCH28080 sensitivity measurements revealed E1P-shifted, ion transport-impaired phenotypes for two N-terminally truncated H,K β-variants, thus substantiating the E2P-stabilizing effect of the β-N terminus suggested by the recent cryo-EM structure.  相似文献   

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Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu297, Glu303, and Asp382) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu297, Glu303, or Asp382 and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp382 seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.It is well known that bovine TSH (bTSH)2 has a higher affinity to the human TSHR (hTSHR) and a 6–10-fold higher intrinsic signaling activity than human TSH (hTSH) (1–5). Human TSH and bTSH share high amino acid sequence identity in the α-subunit (74.1%) and β-subunit (88.4%) (6). Studies involving fusion of hTSH and bTSH α- and β-subunits indicate that the higher affinity and the superagonistic cAMP activity of bTSH at the hTSHR depend primarily on amino acid sequences of the bTSH α-subunit (6). The most noticeable sequence differences between bovine and human TSH consist of four positively charged residues located in the surface-exposed loops of the α-subunit and one positively charged residue in the β-subunit of bTSH (Fig. 1). Moreover, it has previously been shown that positively charged α loop 1 (α-L1) residues are important for the high bioactivity of bTSH, and they have been implicated in receptor binding. These specific characteristics led to the generation of superagonistic hTSH analogs (6). The human TSH analog TR1401 and bTSH differ from hTSH most importantly by four additional positively charged amino acids located in close spatial proximity at the α-L1, of which three are located at identical positions in bTSH and TR1401 (Fig. 1).Open in a separate windowFIGURE 1.Sequence differences between TSH variants used in the present study. A, alignment of the α- and β-subunit of the hTSH (SwissProt: GLHA_HUMAN P01215, TSHB_HUMAN P01222), bTSH (GLHA_BOVIN P01217, TSHB_BOVIN P01223), and the superagonistic hTSH analog TR1401. The additional positively charged residues at TR1401 and at bTSH compared with wt hTSH are boxed in blue. Sequence numbering for human TSH and human analog TR1401 without signal peptide is shown in blue. B, three-dimensional structural TSH models illustrating the spatial localization of the charge related sequence differences between the TSH variants. The TSH α-subunit is shown in gray, and the β-subunit is in orange. Positively charged residues are highlighted in blue, and the C-α atoms of additional positively charged residues compared with hTSH are highlighted by blue globes. Panel i, bovine TSH, characterized by four additional positively charged residues in the α-L1 (T11K, Q13K, P16K, and Q20K) and one positively charged residue in the β-L3 (L69R); panel ii, human TSH without positively charged residues in the α-L1 and β-L3; and panel iii, the human TSH analog TR1401 is characterized by four additional positively charged residues in the α-L1 (Q13K, E14K, P16K, and Q20K) but shows a lack of the additional positively charged residue in the β-L3.TSH binds to the large extracellular region of its receptor. The extracellular region of the TSHR consists of the leucine-rich repeat domain (LRRD), which is linked with the membrane-spanning serpentine domain by the hinge region. Recently, the binding arrangements between the homologous FSH and a part of the FSH receptor ectodomain including the LRRD (FSH receptor amino acids Cys18–Ala246) have been identified (7). However, the hinge region is not contained in this x-ray structure (7).In vitro data provide convincing evidence for the functional importance of the hinge region for receptor activation and TSH binding (8–22). Recently, we specified positions Glu297 and Glu303 in the N-terminal portion and Asp382 in the C-terminal portion of the hTSHR hinge region as important for bTSH binding, suggesting that in the process of bTSH binding an extended hormone-binding site is obviously essential (18). The negative charge of positions Glu297 and Asp382 likely interact with positively charged residues of bTSH by complementary charge-charge interaction (18).To elucidate whether these hinge residues of the hTSHR are specific for interaction with bTSH, we investigated the functional characteristics of the hTSH analog TR1401 and the native ligand hTSH. For the comparison of these two TSH variants with bTSH, we used several mutations and alanine combinations at the signaling and bTSH binding-sensitive hTSHR hinge positions Glu297, Glu303, and Asp382. Our data indicate that the higher bioactivity of the TSH variants TR1401 and bTSH are mediated by specific charged residues of the hormone and the hinge region of the hTSHR. Our findings also support the concept that the hinge region of the TSHR is an modulator of TSH potency and efficacy.  相似文献   

9.
Interleukin-1β (IL-1β) is a master cytokine involved in initiating the innate immune response in vertebrates (Dinarello, C. A. (1994) FASEB J. 8, 1314–1325). It is first synthesized as an inactive 269-residue precursor (pro-interleukin-1β or pro-IL-1β). Pro-IL-1β requires processing by caspase-1 to generate the active, mature 153-residue cytokine. In this study, we combined hydrogen/deuterium exchange mass spectrometry, circular dichroism spectroscopy, and enzymatic digestion comparative studies to investigate the configurational landscape of pro-IL-1β and the role the N terminus plays in modulating the landscape. We find that the N terminus keeps pro-IL-1β in a protease-labile state while maintaining a core region of stability in the C-terminal region, the eventual mature protein. In mature IL-1β, this highly protected region maps back to the area protected earliest in the NMR studies characterizing an on-route kinetic refolding intermediate. This protected region also encompasses two important functional loops that participate in the IL-1β/receptor binding interface required for biological activity. We propose that the purpose of the N-terminal precursor region in pro-IL-1β is to suppress the function of the eventual mature region while keeping a structurally and also functionally important core region primed for the final folding into the native, active state of the mature protein. The presence of the self-inhibiting precursor region provides yet another layer of regulation in the life cycle of this important cytokine.Nearly all cell types respond to interleukin (IL)-1β,4 in a very sensitive manner, via binding to the interleukin-1 receptor type 1 (IL-1RI) (2). Although essential in the immune response, overproduction of IL-1β can lead to both acute (sepsis) as well as chronic (rheumatoid arthritis, atherosclerosis, obesity, and diabetes) disease states (3). Thus, the expression, activation, and secretion of this cytokine are tightly controlled (4). Although many cell types express IL-1β, it is predominately produced and secreted by monocytes and macrophages (1). The protein is synthesized as a biologically inactive 269-residue precursor molecule, pro-interleukin-1β (pro-IL-1β), and the 153-residue active mature IL-1β is generated from the C-terminal domain. Processing of the proprotein involves the recently discovered NALP-1 and NALP-3 inflammasomes, which are responsible for activating procaspase-1 (5). The inflammasome function is integral in wound repair as well as for combating infection (6–9).In vivo, the 31-kDa pro-IL-1β precursor is processed to the active C-terminal 17-kDa form by the interleukin-1 converting enzyme, caspase-1 (10, 11). Caspase-1 is a cysteine protease that recognizes two cleavage sites in pro-IL-1β, the Asp27↓Gly28 and Asp116↓Ala117 peptide bonds (Fig. 1A). These cleavage sites are conserved across mammals (12–14). The activation pathway is believed to proceed with cleavage first at Asp27↓Gly28 (site 1) followed by Asp116↓Ala117 (site 2). These processing events lead to the generation of the mature, active IL-1β from the C-terminal domain of pro-IL-1β (15). After cleavage, the mature protein is exported via a cell-specific non-classical pathway (16). The events leading from caspase-1 activation to active IL-1β secretion are poorly understood and constitute an area of active research (16–20).Open in a separate windowFIGURE 1.A, a schematic of pro-interleukin-1β processing by caspase-1. The two caspase-1 cleavage sites are labeled by residue/number. The products for the cleavage scenario are represented as smaller blocks, and the final mature protein as the actual three-dimensional structure shown in blue (Protein Data Bank code 6I1B (74)). B, panel i, important features are highlighted on the structure of mature IL-1β. Residues Tyr68 (residue 184 in pro-IL-1β) and Trp120 (236 in pro-IL-1β) are indicated by red side chain stick representation. The two loops important for binding at the third Ig domain of the receptor are indicated by blue spheres (the basic/hydrophobic 90s loop, which encompasses residues 85–99 in mature and 201–216 in pro-IL-1β) and yellow spheres (the β-bulge, residues 46–53 and 162–169). The numbering corresponds to mature and pro-IL-1β, respectively. Panel ii, after rotating the structure 90°, the individual trefoils are labeled by color (trefoil 1 in orange, trefoil 2 in yellow, and trefoil 3 in blue). The structural features described in panel i maintain the same coloring. Panel iii, the two-dimensional splay diagram of the trefoils labeled by color as in panel ii showing the 3-fold symmetry of the secondary structure elements.The native structure of IL-1β is classified as a β-trefoil. The global protein-fold contains three pseudo-symmetric βββloopβ motifs that coalesce to form a six-stranded barrel with three hairpins that form a six-stranded cap closing one end of the barrel (see Fig. 1B) (21). Mature IL-1β refolds relatively slowly (22), accessing multiple routes including a major route with a detectable intermediate population (23, 24). Recently, this slow folding has been attributed to repacking of a functionally important loop (the β-bulge) in the mature protein (see Fig. 1B, i) (25–27). Although much information is known about the structure, folding, and function of mature IL-1β, there is little information available on pro-IL-1β, despite the central importance of this molecule in mediating critical inflammatory processes (28–30). What is known is that the presence of the N-terminal 116 amino acids results in a highly protease-sensitive protein with no biological activity (31). Folding of mature IL-1β is believed to occur after cleavage of pro-IL-1β in vivo. Therefore, structural analysis of the precursor is essential for a better understanding of the role the precursor region plays in regulating folding events leading to the generation of the eventual mature protein.The crystal structure of pro-IL-1β has not been determined, despite approximately 25 years of intensive efforts directed toward this goal, as a result of the dynamic nature of this molecule (32–34). Therefore, we used structure-sensitive methods to compare pro-IL-1β in reference to the mature protein. Optical methods in combination with hydrogen/deuterium exchange mass spectrometric analysis (DXMS) and enzymatic digestion were used to investigate how the N-terminal precursor region modulates the properties of the C-terminal mature domain. DXMS is a well established technique for characterizing proteins refractory to standard crystallographic or NMR structure determination techniques (35–37). Taken together, our results indicate that the N terminus inhibits folding to the fully active trefoil structure in the C-terminal region, but maintains the protein in a conformation that is primed for efficient folding upon release after caspase-1 cleavage.  相似文献   

10.
Unsaturated glucuronyl hydrolase (UGL) categorized into the glycoside hydrolase family 88 catalyzes the hydrolytic release of an unsaturated glucuronic acid from glycosaminoglycan disaccharides, which are produced from mammalian extracellular matrices through the β-elimination reaction of polysaccharide lyases. Here, we show enzyme characteristics of pathogenic streptococcal UGLs and structural determinants for the enzyme substrate specificity. The putative genes for UGL and phosphotransferase system for amino sugar, a component of glycosaminoglycans, are assembled into a cluster in the genome of pyogenic and hemolytic streptococci such as Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes, which produce extracellular hyaluronate lyase as a virulent factor. The UGLs of these three streptococci were overexpressed in Escherichia coli cells, purified, and characterized. Streptococcal UGLs degraded unsaturated hyaluronate and chondroitin disaccharides most efficiently at approximately pH 5.5 and 37 °C. Distinct from Bacillus sp. GL1 UGL, streptococcal UGLs preferred sulfated substrates. DNA microarray and Western blotting indicated that the enzyme was constitutively expressed in S. agalactiae cells, although the expression level increased in the presence of glycosaminoglycan. The crystal structure of S. agalactiae UGL (SagUGL) was determined at 1.75 Å resolution by x-ray crystallography. SagUGL adopts α6/α6-barrel structure as a basic scaffold similar to Bacillus UGL, but the arrangement of amino acid residues in the active site differs between the two. SagUGL Arg-236 was found to be one of the residues involved in its activity for the sulfated substrate through structural comparison and site-directed mutagenesis. This is the first report on the structure and function of streptococcal UGLs.Cell surface polysaccharides play an important role in linking neighboring cells and protecting cells against physicochemical stress such as osmotic pressure or invasion by pathogens. Glycosaminoglycans such as chondroitin, hyaluronan, and heparin are highly negatively charged polysaccharides with a repeating disaccharide unit consisting of an uronic acid residue (glucuronic or iduronic acid) and an amino sugar residue (glucosamine or galactosamine) (1), and they are widely present in mammalian cells as an extracellular matrix responsible for cell-to-cell association, cell signaling, and cell growth and differentiation (2). For example, in humans, glycosaminoglycans exist in tissues such as the eye, brain, liver, skin, and blood (3). Except for hyaluronan, glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin sulfate, and heparan sulfate are often sulfated. Chondroitin consists of d-glucuronic acid (GlcA)2 and N-acetyl-d-galactosamine (GalNAc) with a sulfate group(s) at position 4 or 6 or both (4). Hyaluronan, is composed of GlcA and N-acetyl-d-glucosamine (GlcNAc) (5).The adhesion of pathogenic bacteria to mammalian cells is regarded as a primary mechanism of bacterial infection, followed by secondary effects of the infectious process. Polysaccharides, including the glycosaminoglycans that form part of the cell surface matrix, are typical targets for microbial pathogens that invade host cells, and many specific interactions between pathogens and these polysaccharides have been described (6). Glycosaminoglycans in the extracellular matrix are also degraded enzymatically by hydrolases and lyases (1). Generally, hydrolases cleave the glycoside bonds between the glycosyl oxygen and the anomeric carbon atom through the addition of water and play an important role in glycosaminoglycan metabolism in mammals (7). On the other hand, bacterial pathogens invading host cells degrade glycosaminoglycans through the action of lyases. Bacterial polysaccharide lyases recognize the uronic acid residue in polysaccharides, cleave the glycoside bonds through the β-elimination reaction without water addition, and produce unsaturated saccharides with the unsaturated uronic acid residue having a CC double bond at the nonreducing terminus (8).Streptococci such as group B Streptococcus agalactiae, group nonassigned Streptococcus pneumoniae, and group A Streptococcus pyogenes are typical pyogenic and hemolytic pathogens causing severe infections (e.g. pneumonia, bacteremia, sinusitis, or meningitis) (9–11). In S. pneumoniae, hyaluronate lyase, neuraminidases, autolysin, choline-binding protein A, and pneumococcal surface protein A are suggested to function as cell surface virulent factors (12). Hyaluronate lyase degrades the extracellular matrix component hyaluronan in mammalian cells through the β-elimination reaction and releases unsaturated disaccharide, indicating that the enzyme produced by pathogenic bacteria functions as a spreading factor (13). Because hyaluronate lyase is commonly produced by the three pyogenic and hemolytic streptococci (14–16), the structure and function of their enzymes have been intensively studied (17, 18). Groups A, B, C, and G streptococci also produce hyaluronate lyase (19), suggesting that the enzyme is ubiquitously present in pathogenic streptococci. Streptococcal hyaluronate lyase can also act on sulfated and nonsulfated chondroitin (20). The metabolism of the resultant unsaturated disaccharides in streptococci, however, remains to be clarified.Unsaturated glucuronyl hydrolase (UGL), a member of the glycoside hydrolase family 88 in the CAZY data base (21), acts on unsaturated oligosaccharides having an unsaturated GlcA (ΔGlcA) with β-glycoside bond, such as ΔGlcA-GalNAc produced by chondroitin lyase and ΔGlcA-GlcNAc produced by hyaluronate lyase (22) (Fig. 1A). We have first identified the UGL-coding gene in Bacillus sp. GL1 (23) and clarified the structure and function of the enzyme by x-ray crystallography (24–27). The enzyme reaction generates ΔGlcA and the leaving saccharide. ΔGlcA is spontaneously converted to 4-deoxy-1-threo-5-hexosulose-uronate (Fig. 1A) because the ringed form of ΔGlcA has not been obtained because of keto-enole equilibrium (23, 28). In contrast with general glycoside hydrolases with retention or inversion catalytic mechanism of an anomeric configuration, UGL uniquely triggers hydrolysis of vinyl ether groups in unsaturated saccharides but not of the glycoside bond (26) (Fig. 1B). This article deals with the characteristics of streptococcal UGLs by using recombinant enzymes, gene expression in S. agalactiae cells by DNA microarray, and structural determinants of S. agalactiae UGL for substrate specificity by x-ray crystallography and site-directed mutagenesis.Open in a separate windowFIGURE 1.UGL reaction. A, degradation scheme of Δ6S by UGL. B, catalytic reaction mechanism of UGL. C, structures of unsaturated oligosaccharides. ΔGellan, unsaturated gellan tetrasaccharide; ΔHA, unsaturated hyaluronan disaccharide; Δ0S, unsaturated chondroitin disaccharide; Δ2′S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue; Δ2′S4S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-4 position of GalNAc residue; Δ2′S6S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-6 position of GalNAc residue; Δ4S6S, unsaturated chondroitin disaccharide sulfated at C-4 and C-6 positions of GalNAc residue; Δ2′S4S6S, unsaturated chondroitin disaccharide sulfated at C-2 position of ΔGlcA residue and C-4 and C-6 positions of GalNAc residue.  相似文献   

11.
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12.
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13.
Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.Nicotinic acetylcholine receptors (nAChRs)3 are prototypical members of the Cys loop superfamily of neurotransmitter-gated ion channels that mediate the actions of the neurotransmitter acetylcholine (1). nAChRs from vertebrate skeletal muscle and the electric organs of Torpedo rays are heteropentamers of homologous subunits with a stoichiometry of 2α:β:γ(ϵ):δ that are arranged pseudosymmetrically around central cation-selective ion channels (1, 2). There are 12 mammalian neuronal nAChR subunit genes: nine neuronal α subunits (α2–α10) and three neuronal β subunits (β2–β4). The α4β2 nAChR is the most abundant and widely distributed nAChR subtype expressed in the brain and is a major target for potential therapeutic agents for neurological diseases and conditions, including nicotine dependence and Alzheimer and Parkinson diseases (3, 4). Although the ratio of α4 to β2 subunit in vivo is uncertain, expressed receptors containing either three α4 or three β2 subunits have distinct pharmacological properties (5, 6).The agonist binding sites (ABS) of nAChRs are located within the amino-terminal extracellular domain at the interface of adjacent subunits (α-γ and α-δ in the Torpedo nAChR), and different nAChR subunit combinations form ABS with distinct physical and pharmacological properties (3, 7). Affinity labeling studies with Torpedo nAChR and site-directed mutational analyses of muscle and neuronal nAChRs identified key amino acids delineating the ABS from three noncontiguous stretches of the α subunit (Segments A-C, the principal component (+ face)) and three noncontiguous regions of the non-α subunit (Segments D–F, the complementary component (− face)) (8, 9). The three-dimensional structure of the ABS in the absence and presence of nAChR agonists or competitive antagonists has been determined for snail acetylcholine-binding proteins (AChBPs) that are soluble homopentamers homologous to the extracellular (amino-terminal) domain of a nAChR (10–12). In the AChBP, four aromatic amino acids from Segments A–C that are conserved within α subunits, along with a conserved Trp in Segment D, form a core aromatic “pocket” with a dimension optimal for accommodation of a trimethylammonium group. The other amino acids in the non-α subunits closest to the aromatic pocket, which are generally not conserved among γ, δ, or neuronal β subunits, are on three antiparallel β strands. The AChBP structure was used to refine the structure of the Torpedo nAChR in the absence of agonist to 4 Å resolution (13). In this structure, there is a reorientation of Segments A–C, resulting in the absence of a well defined core aromatic binding pocket.Analysis of agonist interactions with mutant nAChRs containing fluorine-substituted core aromatic residues provides evidence that cation-π interactions, particularly with αTrp149 in Segment B, are important determinants of agonist binding affinity (14) and for the higher affinity binding of nicotine to α4β2 nAChRs compared with α2βγδ nAChRs (15). Mutational analyses and molecular docking calculations have also provided evidence that two molecules of very similar structure may actually bind to a single receptor in very different orientations, as seen for two high affinity antagonists, d-tubocurarine and its quaternary ammonium analog metocurine, binding to the AChBP and to the muscle nAChR (16, 17).Photoaffinity labeling provides an alternative means to identify amino acids contributing to a drug binding site (18, 19) and has been used to determine the orientation of drugs bound in the ABS of Torpedo nAChR (20). Epibatidine binds with very high affinity (∼10 pm) to heteromeric neuronal nAChRs (e.g. α4β2) and with nanomolar affinity to α7 and muscle-type/Torpedo nAChRs (3). Utilizing a photoreactive analogue of epibatidine (azidoepibatidine; Fig. 1) and mass spectrometry, Tomizawa et al. (21) identified photolabeled amino acids in the Aplysia AChBP (Tyr195 in Segment C and Met116 in Segment E), establishing an orientation for bound azidoepibatidine consistent with the orientation of epibatidine in an AChBP crystal structure (12).Open in a separate windowFIGURE 1.Structure of [3H]epibatidine (top) and azidoepibatidine (bottom).In this report, we use [3H]epibatidine as a photoaffinity reagent to identify the amino acids photolabeled in an expressed α4β2 nAChR and in the Torpedo α2βγδ nAChR. Comparisons of the labeled amino acids seen in the Torpedo nAChR α-γ binding site and in the α4β2 nAChR, in conjunction with the results of docking calculations for epibatidine binding to homology models of the α2βγδ and α4β2 nAChRs, suggests that epibatidine binds in a single orientation in the α-γ site but in two orientations in the α4β2 ABS.  相似文献   

14.
Ror2 Receptor Requires Tyrosine Kinase Activity to Mediate Wnt5A Signaling   总被引：1，自引：0，他引：1  
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15.
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16.
We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5′ to 3′ direction. A leading strand is synthesized by DNA polymerase ϵ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase δ (pol δ)3 in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ∼150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase α/primase (pol α) initiates each fragment by synthesizing an RNA/DNA primer consisting of ∼1-nt of RNA and ∼10–20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol δ then complexes with PCNA and extends the primer. When pol δ reaches the 5′-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol δ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7–9). FEN1 binds at the 5′-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol δ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol δ and FEN1 also tends to keep flaps short. However, biochemical reconstitution studies demonstrate that some flaps can become long (13, 14). Once these flaps reach ∼30 nt, they can be bound by the eukaryotic single strand binding protein replication protein A (RPA) (15). Binding by RPA to a flap substrate inhibits cleavage by FEN1 (16). The RPA-bound flap would then require another mechanism for proper processing.This second mechanism is proposed to utilize Dna2 (16) in addition to FEN1. Dna2 is both a 5′-3′ helicase and an endonuclease (17, 18). Like FEN1, Dna2 recognizes 5′-flap structures, binding at the 5′-end of the flap and tracking downward toward the base (19, 20). Unlike FEN1, Dna2 cleaves the flap multiple times but not all the way to the base, such that a short flap remains (20). RPA binding to a flap has been shown to stimulate Dna2 cleavage (16). Therefore, if a flap becomes long enough to bind RPA, Dna2 binds and cleaves it to a length of 5–10 nucleotides from which RPA dissociates (21). FEN1 can then enter the flap, displace the Dna2, and then cleave at the base to make the nick for ligation (16, 18, 22). The need for this mechanism may be one reason why DNA2 is an essential gene in Saccharomyces cerevisiae (23, 24). It has been proposed that, in the absence of Dna2, flaps that become long enough to bind RPA cannot be properly processed, leading to genomic instability and cell death (23).In reconstitution of Okazaki fragment processing with purified proteins, even though some flaps became long enough to bind RPA, FEN1 was very effective at cleaving essentially all of the generated flaps (13, 14). Evidently, FEN1 could engage the flaps before binding of RPA. However, these reconstitution assays did not include the 5′-3′ helicase Pif1 (25, 26). Pif1 is involved in telomeric and mitochondrial DNA maintenance (26) and was first implicated in Okazaki fragment processing from genetic studies in S. cerevisiae. Deletion of PIF1 rescued the lethality of dna2Δ, although the double mutant was still temperature-sensitive (27). The authors of this report proposed that Pif1 creates a need for Dna2 by promoting longer flaps. Further supporting this conclusion, deletion of POL32, which encodes the subunit of pol δ that interacts with PCNA, rescued the temperature sensitivity of the dna2Δpif1Δ double mutant (12, 27). Importantly, pol δ exhibited reduced strand displacement activity when POL32 was deleted (12, 28, 29). The combination of pif1Δ and pol32Δ is believed to create a situation in which virtually no long flaps are formed, eliminating the requirement for Dna2 flap cleavage (27).We recently performed reconstitution assays showing that Pif1 can assist in the creation of long flaps. Inclusion of Pif1, in the absence of RPA, increased the proportion of flaps that lengthened to ∼28–32 nt before FEN1 cleavage (14). With the addition of RPA, the appearance of these long flap cleavage products was suppressed. Evidently, Pif1 promoted such rapid flap lengthening that RPA bound some flaps before FEN1 and inhibited cleavage. The RPA-bound flaps would presumably require cleavage by Dna2 for proper processing.Only a small fraction of flaps became long with Pif1. However, there are hundreds of thousands of Okazaki fragments processed per replication cycle (30). Therefore, thousands of flaps are expected to be lengthened by Pif1 in vivo, a number significant enough that improper processing of such flaps could lead to cell death.Our goal here was to determine whether Pif1 can influence the flow of Okazaki fragments through the two proposed pathways. We first questioned whether Pif1 stimulates strand displacement synthesis by pol δ. Next, we asked whether Pif1 lengthens short flaps so that Dna2 can bind and cleave. Finally, we used a complete reconstitution system to determine whether Pif1 promotes creation of RPA-bound flaps that require cleavage by both Dna2 and FEN1 before they can be ligated. Our results suggest that Pif1 promotes the two-nuclease pathway, and reveal the mechanisms involved.  相似文献   

17.
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18.
Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function. Their essential roles in embryo development and tissue function have been demonstrated by numerous genetic studies and the analysis of severe human diseases resulting from mutations in laminin genes (1–4). All laminins are heterotrimers composed of three different gene products, termed α, β, and γ chains. At present, 16 mouse and human laminins are known, assembled from five α, three β, and three γ chains. The different laminins have characteristic expression patterns and functions in the embryo and adult animal (1). Laminins are cross-shaped molecules: the three short arms are composed of one chain each, while the long arm is a coiled coil of all three chains, terminating in a tandem of five laminin G-like (LG)2 domains, LG1-5, contributed by the α chain (2). Basement membrane assembly requires polymerization via the short arms and cell attachment via the LG1-5 region (5, 6).Cell adhesion to laminins is mediated by multiple receptors: integrins bind to the LG1-3 region, whereas α-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids bind predominantly to sites in the LG4-5 pair (7). Integrins are heterodimers with a large extracellular domain consisting of one α and one β chain, which both span the cell membrane and engage in transmembrane signaling (8). Of the 24 mouse and human integrins, the major laminin binding integrins are α3β1, α6β1, α7β1, and α6β4, which have distinct affinities for the different laminin isoforms (9). Although some studies have reported integrin binding or integrin-mediated cell adhesion to isolated LG domains or tandems (10–12), there is strong evidence to suggest that the coiled coil region and an intact γ chain tail are required for full integrin binding to the laminin LG1-3 region (13–18). Compared with integrin binding to collagen and fibronectin, which is understood in atomic detail (19, 20), the laminin-integrin interaction remains poorly characterized in structural terms. We previously determined crystal structures of the LG4-5 region of the laminin α1 and α2 chains and defined their receptor binding sites (21–23). Here, we report the crystal structure of the remainder of the laminin α2 receptor binding region, LG1-3.  相似文献   

19.
We recently reported a critical role of NFκB in mediating hyperproliferative and anti-apoptotic effects of progastrin on proximal colonic crypts of transgenic mice overexpressing progastrin (Fabp-PG mice). We now report activation of β-catenin in colonic crypts of mice in response to chronic (Fabp-PG mice) and acute (wild type FVB/N mice) progastrin stimulation. Significant increases were measured in relative levels of cellular and nuclear β-catenin and pβ-cat45 in proximal colonic crypts of Fabp-PG mice compared with that in wild type littermates. Distal colonic crypts were less responsive. Interestingly, β-catenin activation was downstream of IKKα,β/NFκB, because treatment of Fabp-PG mice with the NFκB essential modulator (NEMO) peptide (inhibitor of IKKα,β/NFκB activation) significantly blocked increases in cellular/nuclear levels of total β-catenin/pβ-cat45/and pβ-cat552 in proximal colons. Cellular levels of pβ-cat33,37,41, however, increased in proximal colons in response to NEMO, probably because of a significant increase in pGSK-3βTyr216, facilitating degradation of β-catenin. NEMO peptide significantly blocked increases in cyclin D1 expression, thereby, abrogating hyperplasia of proximal crypts. Goblet cell hyperplasia in colonic crypts of Fabp-PG mice was abrogated by NEMO treatment, suggesting a cross-talk between the NFκB/β-catenin and Notch pathways. Cellular proliferation and crypt lengths increased significantly in proximal but not distal crypts of FVB/N mice injected with 1 nm progastrin associated with a significant increase in cellular/nuclear levels of total β-catenin and cyclin D1. Thus, intracellular signals, activated in response to acute and chronic stimulation with progastrin, were similar and specific to proximal colons. Our studies suggest a novel possibility that activation of β-catenin, downstream to the IKKα,β/NFκB pathway, may be integral to the hyperproliferative effects of progastrin on proximal colonic crypts.Accumulating evidence suggests that gastrins play an important role in proliferation and carcinogenesis of gastrointestinal and pancreatic cancers (1, 2). Progastrin and glycine-extended gastrin (G-Gly)3 are predominant forms of gastrins found in many tumors, including colon (3–5). Progastrin exerts potent proliferative and anti-apoptotic effects in vitro and in vivo on intestinal mucosal cells (6–10) and on pancreatic cancer cells (11). Transgenic mice overexpressing progastrin from either the liver (hGAS) or intestinal epithelial cells (Fabp-PG) are at a higher risk for developing pre-neoplastic and neoplastic lesions in colons in response to azoxymethane (12–15). Treatment with G-Gly similarly increased the risk for developing pre-neoplastic lesions in rats (16). Thus progastrin and G-Gly exert co-carcinogenic effects in vivo (12–16).Under physiological conditions, only processed forms of gastrins (G17, G34) are present in the circulation (17). In certain disease states, elevated levels of circulating progastrin (0.1 to >1.0 nm) are measured (1). Because co-carcinogenic effects of progastrin are measured in Fabp-PG mice, which express pathophysiological concentrations of hProgastrin (<1–5 nm) (12), elevated levels of circulating progastrin measured in certain disease states in humans may play a role in colon carcinogenesis. A curious finding was that pre-neoplastic and neoplastic lesions were significantly increased in proximal, but not distal, colons of Fabp-PG mice, in response to azoxymethane (12, 14), which may reflect an increase in proliferation and a decrease in azoxymethane-induced apoptosis in proximal colons of Fabp-PG mice (18). We reported a critical role of NFκB activation in mediating proliferation and the anti-apoptotic effect of progastrin on pancreatic cancer cells (in vitro) and on proximal colonic crypts of Fabp-PG mice (in vivo) (11, 18). Whereas the Wnt/β-catenin pathway is known to play a role in the proliferation of colonic crypts (19), its role in mediating biological effects of progastrin remains unknown.β-Catenin is regulated by canonical (GSK-3β phosphorylation-dependent) and non-canonical (GSK-3β phosphorylation-independent) pathways. In the canonical pathway, inhibition of GSK-3β protects β-catenin against degradation by protein complexes, consisting of GSK-3β, axin, and adenomatous polyposis coli (20). In a resting cell, β-catenin is not present in the cytoplasm or nucleus because of proteasomal degradation of β-catenin that is not bound to E-cadherin (20). Following inactivation of GSK-3β, β-catenin stabilizes in the cytoplasm and translocates to the nucleus where it cooperates with Tcf/Lef for activation of target genes (20). In the current studies, we examined whether β-catenin is activated in proximal versus distal colonic crypts of Fabp-PG mice. Relative levels of β-catenin and its target gene product, cyclin D1, were significantly increased in proximal versus distal colonic crypts of Fabp-PG mice. We next examined a possible cross-talk between NFκB and β-catenin activation and the role of GSK-3β. Our results suggest the novel possibility that β-catenin activation in response to progastrin is downstream to IKKα,β/NFκB p65 activation, and that phosphorylation of GSK-3β at Tyr216 may be critically involved.To examine whether differences measured in the response of proximal versus distal colons in Fabp-PG mice were not an artifact of chronic stimulation, we additionally injected WT FVB/N mice with progastrin, as an acute model of stimulation. Our results confirmed that differences we had measured in Fabp-PG mice are not an artifact of chronic stimulation but represent inherent differences in the response of proximal versus distal colonic crypts to circulating progastrins.We and others (18, 21) have previously demonstrated goblet cell hyperplasia in colonic crypts of transgenic mice overexpressing progastrin. In the current studies, we confirmed a significant increase in goblet cell hyperplasia/metaplasia (?) in proximal colonic crypts of Fabp-PG mice. Importantly, goblet cell hyperplasia was reversed to wild type levels by attenuating NFκB activation (and hence β-catenin activation) in NEMO-treated mice. The results of the current studies thus further suggest that pathways which dictate goblet cell lineage may be modulated by progastrin and may be downstream of NFκB/β-catenin activation. This represents a novel paradigm, which needs to be further examined.  相似文献   

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