共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
3.
Chen-Yin Ou Jeong Hoon Kim Catherine K. Yang Michael R. Stallcup 《The Journal of biological chemistry》2009,284(31):20629-20637
4.
Wen Zhang Jun Yang Yajuan Liu Xi Chen Tianxin Yu Jianhang Jia Chunming Liu 《The Journal of biological chemistry》2009,284(34):22649-22656
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 (1–3). 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 (8–10); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (11–21). 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
5.
6.
Daya I. Perkins James R. Trudell Daniel K. Crawford Liana Asatryan Ronald L. Alkana Daryl L. Davies 《The Journal of biological chemistry》2009,284(40):27304-27314
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 (3–5). 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 (6–12). 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, 15–20) 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 (23–25).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 (7–12, 18, 19, 26–30). 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, 34–40).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 (44–51). 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
7.
8.
Michael A. Weiss 《The Journal of biological chemistry》2009,284(29):19159-19163
Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM2 (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM2 (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding. 相似文献
9.
Donghong He Yanlin Su Peter V. Usatyuk Ernst Wm. Spannhake Paul Kogut Julian Solway Viswanathan Natarajan Yutong Zhao 《The Journal of biological chemistry》2009,284(36):24123-24132
Lysophosphatidic acid (LPA), a bioactive phospholipid, induces a wide range of cellular effects, including gene expression, cytoskeletal rearrangement, and cell survival. We have previously shown that LPA stimulates secretion of pro- and anti-inflammatory cytokines in bronchial epithelial cells. This study provides evidence that LPA enhances pulmonary epithelial barrier integrity through protein kinase C (PKC) δ- and ζ-mediated E-cadherin accumulation at cell-cell junctions. Treatment of human bronchial epithelial cells (HBEpCs) with LPA increased transepithelial electrical resistance (TER) by ∼2.0-fold and enhanced accumulation of E-cadherin to the cell-cell junctions through Gαi-coupled LPA receptors. Knockdown of E-cadherin with E-cadherin small interfering RNA or pretreatment with EGTA (0.1 mm) prior to LPA (1 μm) treatment attenuated LPA-induced increases in TER in HBEpCs. Furthermore, LPA induced tyrosine phosphorylation of focal adhesion kinase (FAK) and overexpression of the FAK inhibitor, and FAK-related non-kinase-attenuated LPA induced increases in TER and E-cadherin accumulation at cell-cell junctions. Overexpression of dominant negative protein kinase δ and ζ attenuated LPA-induced phosphorylation of FAK, accumulation of E-cadherin at cell-cell junctions, and an increase in TER. Additionally, lipopolysaccharide decreased TER and induced E-cadherin relocalization from cell-cell junctions to cytoplasm in a dose-dependent fashion, which was restored by LPA post-treatment in HBEpCs. Intratracheal post-treatment with LPA (5 μm) reduced LPS-induced neutrophil influx, protein leak, and E-cadherin shedding in bronchoalveolar lavage fluids in a murine model of acute lung injury. These data suggest a protective role of LPA in airway inflammation and remodeling.The airway epithelium is the site of first contact for inhaled environmental stimuli, functions as a physical barrier to environmental insult, and is an essential part of innate immunity. Epithelial barrier disruption is caused by inhaled allergens, dust, and irritants, resulting in inflammation, bronchoconstriction, and edema as seen in asthma and other respiratory diseases (1–4). Furthermore, increased epithelial permeability also results in para-cellular leakage of large proteins, such as albumin, immunoglobulin G, and polymeric immunoglobulin A, into the airway lumen (5, 6). The epithelial cell-cell junctional complex is composed of tight junctions, adherens junctions, and desmosomes. These adherens junctions play a pivotal role in regulating the activity of the entire junctional complex because the formation of adherens junctions subsequently leads to the formation of other cell-cell junctions (7–9). The major adhesion molecules in the adherens junctions are the cadherins. E-cadherin is a member of the cadherin family that mediates calcium-dependent cell-cell adhesion. The N-terminal ectodomain of E-cadherin contains homophilic interaction specificity, and the cytoplasmic domain binds to catenins, which interact with actin (10–13). Plasma membrane localization of E-cadherin is critical for the maintenance of epithelial cell-cell junctions and airway epithelium integrity (7, 10, 14). A decrease of adhesive properties of E-cadherin is related to the loss of differentiation and the subsequent acquisition of a higher motility and invasiveness of epithelial cells (10, 14, 15). Dislocation or shedding of E-cadherin in the airway epithelium induces epithelial shedding and increases airway permeability in lung airway diseases (10, 14, 16). In an ovalbumin-challenged guinea pig model of asthma, it has been demonstrated that E-cadherin is dislocated from the lateral margins of epithelial cells (10). Histamine increases airway para-cellular permeability and results in an increased susceptibility of airway epithelial cells to adenovirus infection by interrupting E-cadherin adhesion (14). Serine phosphorylation of E-cadherin by casein kinase II, GSK-3β, and PKD1/PKC2 μ enhanced E-cadherin-mediated cell-cell adhesion in NIH3T3 fibroblasts and LNCaP prostate cancer cells (11, 17). However, the regulation and mechanism by which E-cadherin is localized within the pulmonary epithelium is not fully known, particularly during airway remodeling.LPA, a naturally occurring bioactive lipid, is present in body fluids, such as plasma, saliva, follicular fluid, malignant effusions, and bronchoalveolar lavage (BAL) fluids (18–20). Six distinct high affinity cell-surface LPA receptors, LPA-R1–6, have been cloned and described in mammals (21–26). Extracellular activities of LPA include cell proliferation, motility, and cell survival (27–30). LPA exhibits a wide range of effects on differing cell types, including pulmonary epithelial, smooth muscle, fibroblasts, and T cells (31–35). LPA augments migration and cytokine synthesis in lymphocytes and induces chemotaxis of Jurkat T cells through Matrigel membranes (34). LPA induces airway smooth muscle cell contractility, proliferation, and airway repair and remodeling (35, 36). LPA also potently stimulates IL-8 (31, 37–39), IL-13 receptor α2 (IL-13Rα2) (40), and COX-2 gene expression and prostaglandin E2 release (41) in HBEpCs. Prostaglandin E2 and IL-13Rα2 have anti-inflammatory properties in pulmonary inflammation (42, 43). These results suggest that LPA may play a protective role in lung disease by stimulating an innate immune response while simultaneously attenuating the adaptive immune response. Furthermore, intravenous injection with LPA attenuated bacterial endotoxin-induced plasma tumor necrosis factor-α production and myeloperoxidase activity in the lungs of mice (44), suggesting an anti-inflammatory role for LPA in a murine model of sepsis.We have reported that LPA induces E-cadherin/c-Met accumulation in cell-cell contacts and increases TER in HBEpCs (45). Here, for the first time, we report that LPA-induced increases in TER are dependent on PKCδ, PKCζ, and FAK-mediated E-cadherin accumulation at cell-cell junctions. Furthermore, we demonstrate that post-treatment of LPA rescues LPS-induced airway epithelial disruption in vitro and reduces E-cadherin shedding in a murine model of ALI. This study identifies the molecular mechanisms linking the LPA and LPA receptors to maintaining normal pulmonary epithelium barrier function, which is critical in developing novel therapies directed at ameliorating pulmonary diseases. 相似文献
10.
Ya Hui Hung Elysia L. Robb Irene Volitakis Michael Ho Genevieve Evin Qiao-Xin Li Janetta G. Culvenor Colin L. Masters Robert A. Cherny Ashley I. Bush 《The Journal of biological chemistry》2009,284(33):21899-21907
Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H2O2 and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).2 Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPPβ and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu2+ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H2O2 and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu2+ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu2+ under copper-deficiency conditions to form Aβ·Cu2+ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts. 相似文献
11.
Qin Wang Dhaarmini Rajshankar Donald R. Branch Katherine A. Siminovitch Maria Teresa Herrera Abreu Gregory P. Downey Christopher A. McCulloch 《The Journal of biological chemistry》2009,284(31):20763-20772
Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) α in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPα to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R. In response to IL-1, catalytically active PTPα was required for Ca2+ release from the ER, Src-dependent phosphorylation of IP3R1 and accumulation of IP3R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPα was required for the association of PTPα with IP3R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPα acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca2+ signaling.The interleukin-1 (IL-1)3 family of pro-inflammatory cytokines mediates host responses to infection and injury. Impaired control of IL-1 signaling leads to chronic inflammation and destruction of extracellular matrices (1, 2), as seen in pathological conditions such as pulmonary fibrosis (3), rheumatoid arthritis (4, 5), and periodontitis (6). IL-1 elicits multiple signaling programs, some of which trigger Ca2+ release from the endoplasmic reticulum (ER) as well as expression of multiple cytokines and inflammatory factors including c-Fos and c-Jun (7, 8), and matrix metalloproteinases (9, 10), which mediate extracellular matrix degradation via mitogen-activated protein kinase-regulated pathways (11).In anchorage-dependent cells including fibroblasts and chondrocytes, focal adhesions (FAs) are required for IL-1-induced Ca2+ release from the ER and activation of ERK (12–14). FAs are actin-enriched adhesive domains composed of numerous (>50) scaffolding and signaling proteins (15–17). Many FA proteins are tyrosine-phosphorylated, including paxillin, focal adhesion kinase, and src family kinases, all of which are crucial for the assembly and disassembly of FAs (18–21). Protein-tyrosine phosphorylation plays a central role in regulating many cellular processes including adhesion (22, 23), motility (24), survival (25), and signal transduction (26–29). Phosphorylation of proteins by kinases is balanced by protein-tyrosine phosphatases (PTP), which can enhance or attenuate downstream signaling by dephosphorylation of tyrosine residues (30–32).PTPs can be divided into two main categories: receptor-like and intracellular PTPs (33). Two receptor-like PTPs have been localized to FA (leukocyte common antigen-related molecule and PTPα). Leukocyte common antigen-related molecule can dephosphorylate and mediate degradation of p130cas, which ultimately leads to cell death (34, 35). PTPα contains a heavily glycosylated extracellular domain, a transmembrane domain, and two intracellular phosphatase domains (33, 36). The amino-terminal domain predominantly mediates catalytic activity, whereas the carboxyl-terminal domain serves a regulatory function (37, 38). PTPα is enriched in FA (23) and is instrumental in regulating FA dynamics (39) via activation of c-Src/Fyn kinases by dephosphorylating the inhibitory carboxyl tyrosine residue, namely Tyr529 (22, 40–42) and facilitation of integrin-dependent assembly of Src-FAK and Fyn-FAK complexes that regulate cell motility (43). Although PTPα has been implicated in formation and remodeling of FAs (44, 45), the role of PTPα in FA-dependent signaling is not defined.Ca2+ release from the ER is a critical step in integrin-dependent IL-1 signal transduction and is required for downstream activation of ERK (13, 46). The release of Ca2+ from the ER depends on the inositol 1,4,5-triphosphate receptor (IP3R), which is an IP3-gated Ca2+ channel (47). All of the IP3R subtypes (subtypes 1–3) have been localized to the ER, as well as other the plasma membrane and other endomembranes (48–50). Further, IP3R may associate with FAs, enabling the anchorage of the ER to FAs (51, 52). However, the molecule(s) that provide the structural link for this association has not been defined.FA-restricted, IL-1-triggered signal transduction in anchorage-dependent cells may rely on interacting proteins that are enriched in FAs and the ER (53). Here, we examined the possibility that PTPα associates with c-Src and IP3R to functionally link FAs to the ER, thereby enabling IL-1 signal transduction. 相似文献
12.
13.
Human T-Cell Leukemia Virus Type 1 Tax Induction of NF-κB Involves Activation of the IκB Kinase α (IKKα) and IKKβ Cellular Kinases
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Romas Geleziunas Sharon Ferrell Xin Lin Yajun Mu Emmett T. Cunningham Jr. Mark Grant Margery A. Connelly John E. Hambor Kenneth B. Marcu Warner C. Greene 《Molecular and cellular biology》1998,18(9):5157-5165
14.
��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*
Jennifer R. Mastroianni Andr�� J. Ouellette 《The Journal of biological chemistry》2009,284(41):27848-27856
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 (1–5). 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 (8–10). 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 (14–18) 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 (27–30). 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
15.
16.
Human T-Cell Leukemia Virus Type 1 Tax Protein Abrogates Interleukin-2 Dependence in a Mouse T-Cell Line 总被引:5,自引:3,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Youichi Iwanaga Tomonori Tsukahara Takashi Ohashi Yuetsu Tanaka Masaaki Arai Masataka Nakamura Kiyoshi Ohtani Yoshihiro Koya Mari Kannagi Naoki Yamamoto Masahiro Fujii 《Journal of virology》1999,73(2):1271-1277
17.
18.
19.
Laszlo L. P. Hosszu Clare R. Trevitt Samantha Jones Mark Batchelor David J. Scott Graham S. Jackson John Collinge Jonathan P. Waltho Anthony R. Clarke 《The Journal of biological chemistry》2009,284(33):21981-21990
Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)2 (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrPSc, differs from the normal cellular form (PrPC) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrPC, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrPC to PrPSc. The most coherent and general model proposed thus far is that PrPC fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrPSc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrPSc-dependent conversion reactions (18–20) or through conversion of PrPC in the absence of a PrPSc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrPSc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrPC and PrPSc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrPSc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrPSc (but not PrPC) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrPSc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP. 相似文献
20.
Rapha?lle Pardossi-Piquard Seung-Pil Yang Soshi Kanemoto Yongjun Gu Fusheng Chen Christopher B?hm Jean Sevalle Tong Li Philip C. Wong Frédéric Checler Gerold Schmitt-Ulms Peter St. George-Hyslop Paul E. Fraser 《The Journal of biological chemistry》2009,284(24):16298-16307
Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.The anterior pharynx defective-1 (APH1)5 protein is an essential component of presenilin-dependent complexes required for the γ/ϵ-secretase activity (1). The multicomponent γ-secretase is responsible for the intramembrane proteolysis of a variety of substrates including the amyloid-β precursor protein (APP) and Notch receptor. Notch signaling is involved in a variety of important cell fate decisions during embryogenesis and adulthood (2). The γ/ϵ-secretase cleavage of APP protein is related to the pathogenesis of Alzheimer disease by releasing the 4-kDa amyloid β-peptide (Aβ) which accumulates as senile plaques in patients with Alzheimer disease (3, 4).The γ-complexes are composed of multispanning transmembrane proteins that include APH1 (5, 6), presenilin (PS1 or PS2) (7–10), PEN2 (5), and the type 1 transmembrane nicastrin (NCT) (11). All four components are essential for proteolytic activity, and loss of any single component destabilizes the complex, resulting in the loss of substrate cleavage. Conversely, co-expression of all four components increases γ-secretase activity (12–14). During the maturation of the complexes, presenilins undergo an endoproteolytic cleavage to generate amino- and carboxyl-terminal fragments which remain associated as heterodimers in the active high molecular weight complexes (15–18). Although the exact function of presenilins has been debated (19, 20), it has been proposed that the presenilins are aspartyl proteases with two transmembrane residues constituting the catalytic subunit (21). Analogous aspartyl catalytic dyads are found in the signal peptide peptidases (21, 22). Contributions from the other components are under investigation, and it has been shown, for example, that the large ectodomain of NCT plays a key role in substrate recognition (23, 24). It has also been shown that other proteins can regulate activity such as TMP21, a member of p24 cargo protein, which binds to the presenilin complexes and selectively modulates γ but not ϵ cleavage (25, 26).APH1 is a seven-transmembrane protein with a topology such that the amino terminus is oriented with the endoplasmic reticulum and the carboxyl terminus resides in the cytoplasm (6, 27). It is also expressed as different isoforms encoded by two genes in humans (APH1a on chromosome 1; APH1b on chromosome 15) or three genes in rodents (APH1a on chromosome 3; APH1b and APH1c on chromosome 9). APH1a has 55% sequence similarity with APH1b/APH1c, whereas APH1b and APH1c share 95% similarity. In addition to these different genes, APH1a is alternatively spliced to generate a short (APH1aS) and a long isoform (APH1aL). These two isoforms differ by the addition of 18 residues on the carboxyl-terminal part of APH1aL (28, 29). Deletion of APH1a in mice is embryonically lethal and is associated with developmental and patterning defects similar to those found in Notch, NCT, or PS1 null embryos (30, 31). In contrast to the essential nature of APH1a, the combined APH1b/c-deficient mice survive into adulthood (31). This suggests that APH1a is the major homologue involved in presenilin-dependent function during embryonic development. In addition, these different APH1 variants are constituents of distinct, proteolytically active presenilin-containing complexes and may, therefore, make unique contributions to γ-secretase activity (30–32).Despite their importance to complex formation and function, the exact role of the APH1 isoforms in presenilin-dependent γ/ϵ-secretase activity remains under investigation. In the current study, several highly conserved polar and charged residues located within the transmembrane domains of APH1 were identified. Mutagenesis of two conserved histidine residues embedded in TM5 and TM6 (His-171 and His-197) lead to alterations in γ-secretase complex maturation and activity. The histidine residues contribute to APH1 function and are involved in stabilizing interactions with other γ-secretase components. These key histidines may also be physically localized near the presenilin active site and involved in the γ-secretase activity as shown by the decreased activity of γ-secretase complexes that are assembled with the His-mutants. 相似文献