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1.
J Biol Chem ; 289(25): 17830-42, 2014 Jun 20.
Article in English | MEDLINE | ID: mdl-24798333

ABSTRACT

Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys(27) to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys(27) reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys(27), pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys(27) precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys(27) variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.


Subject(s)
Calnexin/metabolism , Endoplasmic Reticulum-Associated Degradation/physiology , Polysaccharides/metabolism , Protein Folding , Proteolysis , Receptors, Opioid, delta/metabolism , HEK293 Cells , Humans , Polysaccharides/genetics , Protein Structure, Tertiary , Receptors, Opioid, delta/genetics , Ubiquitination/physiology
2.
Toxicol In Vitro ; 27(5): 1550-61, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23583641

ABSTRACT

Ambient air particulate matter (PM) as well as microbial contaminants in the indoor air are known to cause severe adverse health effects. It has been shown that there is a clear seasonal variation in the potency of outdoor air particles to evoke inflammation and cytotoxicity. However, the role of outdoor sources in the indoor air quality, especially on its toxicological properties, remains largely unknown. In this study, we collected size segregated (PM10-2.5, PM2.5-0.2 and PM0.2) particulate samples with a high volume cascade impactor (HVCI) on polyurethane foam and fluoropore membrane filters. The samples were collected during four different seasons simultaneously from indoor and outdoor air. Thereafter, the samples were weighed and extracted with methanol from the filters before undergoing toxicological analyses. Mouse macrophages (RAW264.7) were exposed to particulate sample doses of 50, 150 and 300µg/ml for 24h. Thereafter, the levels of the proinflammatory cytokine (TNF-α), NO-production, cytotoxicity (MTT-test) and changes in the cell cycle (SubG1, G1, S and G2/M phases) were investigated. PM10-2.5 particles evoked the highest inflammatory and cytotoxic responses. Instead, PM2.5-0.2 samples exerted the greatest effect on apoptotic activity in the macrophages. With respect to the outdoor air samples, particles collected during warm seasons had a stronger potency to induce inflammatory and cytotoxic responses, whereas no such clear effect was seen with the corresponding indoor air samples. Outdoor air samples were associated with higher inflammatory potential, whereas indoor air samples had overall higher cytotoxic properties. This indicates that the outdoor air has a limited influence on the indoor air quality in a modern house. Thus, the indoor sources dominate the toxicological responses obtained from samples collected inside house.


Subject(s)
Air Pollution, Indoor/adverse effects , Particulate Matter/toxicity , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line , Cell Membrane Permeability/drug effects , Cell Survival/drug effects , Cytokines/metabolism , Housing , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Nitric Oxide/metabolism , Particle Size , Particulate Matter/chemistry , Seasons
3.
Toxicol Lett ; 188(3): 214-22, 2009 Aug 10.
Article in English | MEDLINE | ID: mdl-19397966

ABSTRACT

Benzo(a)pyrene (BP) forms benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in human breast adenocarcinoma MCF-7 cells, leading to p53 protein induction and phosphorylation. Although BP-induced apoptosis in rodent cells is known, it is still unclear in human cells. Here we have analyzed the effects of BP on p53 related apoptotic proteins, cell cycle and cell death in MCF-7 cells. PUMA-protein (p53 up-regulated modulator of apoptosis) levels were changed after BP exposure so that PUMA-alpha protein was statistically significantly increased whereas PUMA-beta protein was statistically significantly decreased. PUMA-protein levels were also investigated in ZR-75-1 cells, where PUMA-alpha protein was statistically significantly increased. Cytochrome c, which is released from mitochondria during apoptosis to form the apoptosome, was increased in cytoplasmic fraction after BP exposure in MCF-7 cells. Increased apoptosis was also seen after 48 and 72 h BP exposure (2.5 and 5 microM). In addition, BP decreased dose dependently cell viability (2.5 and 5 microM) and increased ROS formation (1 and 10 microM). Our results suggest that PUMA-alpha protein is involved in BP-induced cell death most likely through a p53 dependent apoptotic pathway.


Subject(s)
Apoptosis Regulatory Proteins/biosynthesis , Apoptosis/drug effects , Benzo(a)pyrene/toxicity , Proto-Oncogene Proteins/biosynthesis , Cell Line, Tumor , Cytochromes c/metabolism , Cytosol/drug effects , Cytosol/metabolism , Down-Regulation , Flow Cytometry , Humans , Immunoblotting , Membrane Potentials/drug effects , Microscopy, Confocal , Mitochondria/drug effects , Mitochondria/metabolism , Protein Isoforms , Reactive Oxygen Species/metabolism , Up-Regulation
4.
Traffic ; 10(1): 116-29, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19000170

ABSTRACT

The human delta opioid receptor (hdeltaOR) is a G-protein-coupled receptor that is mainly involved in the modulation of pain and mood. Only one nonsynonymous single nucleotide polymorphism (T80G) has been described, causing Phe27Cys substitution in the receptor N-terminus and showing association with substance dependence. In this study, we expressed the two hdeltaOR variants in a heterologous expression system with an identical genetic background. They differed greatly during early steps of biosynthesis, displaying a significant difference in the maturation efficiency (50% and 85% for the Cys27 and Phe27 variants, respectively). The Cys27 variant also showed accumulation in pre-Golgi compartments of the secretory pathway and impaired targeting to endoplasmic reticulum (ER)-associated degradation following long-term expression. In addition, the cell surface receptors of the Cys27 variant internalized constitutively. Replacement of phenylalanine with other amino acids revealed that cysteine at position 27 decreased the mature receptor/precursor ratio most extensively, suggesting a thiol-mediated retention of precursors in the ER. However, cysteine did not cause a major folding defect because pharmacological characteristics and the maturation kinetics of the variants were identical, and an opioid antagonist was able to enhance the maturation of both variants. We conclude that, instead of causing loss of function, Phe27Cys polymorphism of the hdeltaOR causes a gain-of-function phenotype, which may have implications for the regulation of receptor expression at the cell surface and possibly also for the susceptibility to pathophysiological states.


Subject(s)
Polymorphism, Genetic/genetics , Protein Processing, Post-Translational/genetics , Receptors, Opioid, delta/metabolism , Amino Acid Sequence , Animals , Cell Line , Conserved Sequence , Cysteine/genetics , Cysteine/metabolism , Endoplasmic Reticulum/metabolism , Gene Expression Regulation , Humans , Kinetics , Molecular Sequence Data , Phenylalanine/genetics , Phenylalanine/metabolism , Protein Transport , Receptors, Opioid, delta/chemistry , Receptors, Opioid, delta/genetics , Sequence Alignment
5.
J Biol Chem ; 283(43): 29086-98, 2008 Oct 24.
Article in English | MEDLINE | ID: mdl-18703511

ABSTRACT

A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn(18) and Asn(33)) of the human delta-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn(33) was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the delta-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.


Subject(s)
Endoplasmic Reticulum/metabolism , Gene Expression Regulation , Polysaccharides/chemistry , Receptors, Opioid, delta/metabolism , Animals , Binding Sites , CHO Cells , Cell Membrane/metabolism , Cricetinae , Cricetulus , Glycosylation , Humans , Kinetics , Ligands , Models, Biological
6.
J Mol Biol ; 371(3): 622-38, 2007 Aug 17.
Article in English | MEDLINE | ID: mdl-17588601

ABSTRACT

Calcium (Ca(2+)) plays a pivotal role in both cellular signaling and protein synthesis. However, it is not well understood how calcium metabolism and synthesis of secreted and membrane-bound proteins are related. Here we demonstrate that the sarco(endo)plasmic reticulum Ca(2+) ATPase 2b (SERCA2b), which maintains high Ca(2+) concentration in the lumen of the endoplasmic reticulum, interacts specifically with the human delta opioid receptor during early steps of receptor biogenesis in human embryonic kidney 293 cells. The interaction involves newly synthesized incompletely folded receptor precursors, because the association between the delta opioid receptor and SERCA2b (i) was short-lived and took place soon after receptor translation, (ii) was not affected by misfolding of the receptor, and (iii) decreased if receptor folding was enhanced by opioid receptor pharmacological chaperone. The physical association with SERCA2b was found to be a universal feature among G protein-coupled receptors within family A and was shown to occur also between the endogenously expressed luteinizing hormone receptor and SERCA2b in rat ovaries. Importantly, active SERCA2b rather than undisturbed Ca(2+) homeostasis was found to be essential for delta opioid receptor biogenesis, as inhibition of its Ca(2+) pumping activity by thapsigargin reduced the interaction and impaired the efficiency of receptor maturation, two phenomena that were not affected by a Ca(2+) ionophore A23187. Nevertheless, inhibition of SERCA2b did not compromise the functionality of receptors that were able to mature. Thus, we propose that the association with SERCA2b is required for efficient folding and/or membrane integration of G protein-coupled receptors.


Subject(s)
Endoplasmic Reticulum/enzymology , Receptors, Opioid, delta/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Animals , Calnexin/metabolism , Cell Line , Endoplasmic Reticulum/drug effects , Enzyme Activation/drug effects , Female , Humans , Immunoprecipitation , Ovary/drug effects , Ovary/enzymology , Pregnancy , Protein Binding/drug effects , Protein Folding , Protein Precursors/metabolism , Rats , Receptors, LH/metabolism , Receptors, Opioid, delta/chemistry , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Substrate Specificity/drug effects , Thapsigargin/pharmacology
7.
J Biol Chem ; 282(32): 23171-83, 2007 Aug 10.
Article in English | MEDLINE | ID: mdl-17550902

ABSTRACT

Accumulating evidence has indicated that membrane-permeable G protein-coupled receptor ligands can enhance cell surface targeting of their cognate wild-type and mutant receptors. This pharmacological chaperoning was thought to result from ligand-mediated stabilization of immature receptors in the endoplasmic reticulum (ER). In the present study, we directly tested this hypothesis using wild-type and mutant forms of the human delta-opioid receptor as models. ER-localized receptors were isolated by expressing the receptors in HEK293 cells under tightly controlled tetracycline induction and blocking their ER export with brefeldin A. The ER-retained delta-opioid receptor precursors were able to bind [(3)H]diprenorphine with high affinity, and treatment of cells with an opioid antagonist naltrexone led to a 2-fold increase in the number of binding sites. After removing the transport block, the antagonist-mediated increase in the number of receptors was detectable at the cell surface by flow cytometry and cell surface biotinylation assay. Importantly, opioid ligands, both antagonists and agonists, were found to stabilize the ER-retained receptor precursors in an in vitro heat inactivation assay and the treatment enhanced dissociation of receptor precursors from the molecular chaperone calnexin. Thus, we conclude that pharmacological chaperones facilitate plasma membrane targeting of delta-opioid receptors by binding and stabilizing receptor precursors, thereby promoting their release from the stringent ER quality control.


Subject(s)
Endoplasmic Reticulum/metabolism , Receptors, Opioid/metabolism , Binding Sites , Biotinylation , Brefeldin A/chemistry , Calnexin/chemistry , Cell Line , Cell Membrane/metabolism , DNA/chemistry , Humans , Kinetics , Ligands , Molecular Chaperones/metabolism , Protein Binding , Receptors, Opioid/chemistry , Receptors, Opioid, delta/chemistry
8.
J Biol Chem ; 281(23): 15780-9, 2006 Jun 09.
Article in English | MEDLINE | ID: mdl-16595649

ABSTRACT

Protein palmitoylation is a reversible lipid modification that plays important roles for many proteins involved in signal transduction, but relatively little is known about the regulation of this modification and the cellular location where it occurs. We demonstrate that the human delta opioid receptor is palmitoylated at two distinct cellular locations in human embryonic kidney 293 cells and undergoes dynamic regulation at one of these sites. Although palmitoylation could be readily observed for the mature receptor (Mr 55,000), [3H]palmitate incorporation into the receptor precursor (Mr 45,000) could be detected only following transport blockade with brefeldin A, nocodazole, and monensin, indicating that the modification occurs initially during or shortly after export from the endoplasmic reticulum. Blocking of palmitoylation with 2-bromopalmitate inhibited receptor cell surface expression, indicating that it is needed for efficient intracellular transport. However, cell surface biotinylation experiments showed that receptors can also be palmitoylated once they have reached the plasma membrane. At this location, palmitoylation is regulated in a receptor activation-dependent manner, as was indicated by the opioid agonist-promoted increase in the turnover of receptor-bound palmitate. This agonist-mediated effect did not require receptor-G protein coupling and occurred at the cell surface without the need for internalization or recycling. The activation-dependent modulation of receptor palmitoylation may thus contribute to the regulation of receptor function at the plasma membrane.


Subject(s)
Palmitic Acid/metabolism , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/metabolism , Subcellular Fractions/metabolism , Cell Line , Humans
9.
Am J Physiol Lung Cell Mol Physiol ; 288(5): L997-1001, 2005 May.
Article in English | MEDLINE | ID: mdl-15626747

ABSTRACT

Peroxiredoxins (Prxs) are a group of thiol containing proteins that participate both in signal transduction and in the breakdown of hydrogen peroxide (H(2)O(2)) during oxidative stress. Six distinct Prxs have been characterized in human cells (Prxs I-VI). Prxs I-IV form dimers held together by disulfide bonds, Prx V forms intramolecular bond, but the mechanism of Prx VI, so-called 1-Cys Prx, is still unclear. Here we describe the regulation of all six Prxs in cultured human lung A549 and BEAS-2B cells. The cells were exposed to variable concentrations of H(2)O(2), menadione, tumor necrosis factor-alpha or transforming growth factor-beta. To evoke glutathione depletion, the cells were furthermore treated with buthionine sulfoximine. Only high concentrations (300 microM) of H(2)O(2) caused a minor increase (<28%, 4 h) in the expression of Prxs I, IV, and VI. Severe oxidant stress (250-500 microM H(2)O(2)) caused a significant increase in the proportion of the monomeric forms of Prxs I-IV; this was reversible at lower H(2)O(2) concentrations (< or =250 microM). This recovery of Prx overoxidation differed among the various Prxs; Prx I was recovered within 24 h, but recovery required 48 h for Prx III. Overall, Prxs are not significantly modulated by mild oxidant stress or cytokines, but there is variable, though reversible, overoxidation in these proteins during severe oxidant exposure.


Subject(s)
Oxidative Stress/physiology , Peroxidases/metabolism , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , Antioxidants/metabolism , Cell Division/physiology , Cell Survival/physiology , Cells, Cultured , Humans , Hydrogen Peroxide/pharmacology , Oxidants/pharmacology , Oxidation-Reduction , Oxidative Stress/drug effects , Peroxiredoxin VI , Peroxiredoxins
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