Involvement of the carboxyl-terminal propeptide of beta-glucuronidase in its compartmentalization within the endoplasmic reticulum as determined by a synthetic peptide approach

The proenzyme form of beta-glucuronidase is compartmentalized in large quantities within the endoplasmic reticulum by binding to the esterase, egasyn. Also, the propeptide of the proenzyme form of beta-glucuronidase is likely located at the carboxyl terminus. We have, therefore, tested if this carboxyl-terminal peptide is important in binding to egasyn. A polyclonal antibody to a 30-mer synthetic peptide, corresponding to the carboxyl-terminal 30 amino acids of pro-beta-glucuronidase, provided evidence that egasyn binds to the carboxyl terminus of beta-glucuronidase. This antibody interacted with proenzyme beta-glucuronidase-egasyn complexes in which one, two, or three egasyn molecules were bound to the beta-glucuronidase tetramer, but not with those complexes (M4) which contained four egasyn molecules. We interpret these results as indicating that all available carboxyl termini of the beta-glucuronidase proenzyme tetramer are shielded by egasyn in the M4 complexes. The same antibody did not recognize the mature lysosomal form of beta-glucuronidase, indicating that only the proenzyme form of microsomal beta-glucuronidase contains the original carboxyl terminus. Also, the synthetic 30-mer was found to be a specific and potent inhibitor (50% inhibition at 1.3 microM) of the esterase activity of purified egasyn but exhibited little inhibitory activity toward other purified esterases including a rat trifluoroacetylated esterase or egasyn esterase from another species. Together, these data describe a potent interaction of the exposed carboxyl terminus of precursor glucuronidase with the esterase catalytic site of egasyn, which in turn results in the specific localization of glucuronidase within the lumen of the endoplasmic reticulum.     

Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.     

Calnexin regulates mammalian Kv1 channel trafficking

Voltage-gated Kv1 channels are key factors regulating excitability in the mammalian central nervous system. Diverse posttranslational regulatory mechanisms operate to determine the density, subunit composition, and localization of Kv1 channel complexes in the neuronal plasma membrane. In this study, we investigated the role of the endoplasmic reticulum chaperone calnexin in the intracellular trafficking of Kv1 channels. We found that coexpressing calnexin with the Kv1.2alpha subunit in transfected mammalian COS-1 cells produced a dramatic dose-dependent increase in cell surface Kv1.2 channel complexes. In calnexin-transfected COS-1 cells, the proportion of Kv1.2 channels with mature N-linked oligosaccharide chains was comparable to that observed in neurons. In contrast, calnexin coexpression exerted no effects on trafficking of the intracellularly retained Kv1.1 or Kv1.6alpha subunits. We also found that calnexin and auxiliary Kvbeta2 subunit coexpression was epistatic, suggesting that they share a common pathway for promoting Kv1.2 channel surface expression. These results provide yet another component in the elaborate repertoire of determinants regulating the density of Kv1 channels in the plasma membrane.     

Relationships between levels of membrane-bound glucuronidase and the associated protein egasyn in mouse tissues

Mouse beta-glucuronidase has a dual intracellular localization, being present in both endoplasmic reticulum and lysosomes of several tissues. Previous studies demonstrated that the protein egasyn is complexed with microsomal but not lysosomal glucuronidase and that a mutant lacking egasyn is deficient in microsomal, but not lysosomal, glucuronidase. By means of a recently developed radioimmunoassay for egasyn, the relationship between microsomal glucuronidase levels and egasyn levels has been examined in various adult tissues, during postnatal development in liver, and after androgen induction of glucuronidase in kidney. The results indicate that the relative availability of egasyn determines the balance between glucuronidase incorporation into membranes and that into lysosomes.     

The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase

Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non-isotopic and radioactive assays. Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis.     

Ca(2+) signals in Pmr1-GFP-expressing COS-1 cells with functional endoplasmic reticulum

We studied the role of the Pmr1-containing Ca(2+) store in COS-1 cells endowed with a functional endoplasmic reticulum. Transfected cells could be recognized by using a green-fluorescent-protein (GFP)-tagged form of Pmr1. Pmr1-GFP fluorescence showed a typical juxtanuclear Golgi-like distribution. Pmr1-GFP-containing cells with functional endoplasmic reticulum responded to 100 microM ATP with baseline Ca(2+) spiking, while non-transfected cells produced an initial Ca(2+) peak followed by a long-lasting plateau. The Ca(2+) signal often appeared after a long latency in Pmr1-GFP-expressing cells. ATP-stimulated Pmr1-GFP-expressing cells with functional endoplasmic reticulum responded after a latency period to extracellular Ca(2+) with a regenerative Ca(2+) signal, while non-transfected control cells responded with an immediate slow rise in free cytosolic Ca(2+) concentration. These results demonstrate the importance of the Pmr1-containing Ca(2+) store in generating or modifying cellular Ca(2+) signals.     

BAP31 is involved in the retention of cytochrome P450 2C2 in the endoplasmic reticulum

Microsomal cytochrome P450 2C2 is an integral endoplasmic reticulum (ER) membrane protein that is directly retained in the ER and excluded from transport vesicles. We have used bimolecular fluorescence complementation and co-immunoprecipitation to show that a ubiquitous ER membrane protein (BAP31) interacts with P450 2C2 in transfected COS-1 cells. A chimera containing only the N-terminal signal anchor of P450 2C1 (P450 2C1-(1-29)) also interacted with BAP31, which is consistent with interaction of the two proteins via their transmembrane domains. Down-regulation of BAP31 expression with small interfering RNA resulted in redistribution of green fluorescent protein-tagged P450 2C2 or P450 2C1-(1-29) from the ER into the nuclear membrane and compact perinuclear compartment structures as well as the cell surface in a small fraction of the cells. In Bap31-null embryonic stem cells, a significant fraction of P450 2C2 or P450 2C1-(1-29) was detected at the cell surface and nuclear envelope, but was redistributed to the ER by expression of BAP31. The expression level of P450 2C2 was significantly increased in COS-1 cells with repressed levels of BAP31. Formation of the pro-apoptotic p20 fragment of BAP31 was detected in transfected COS-1 cells expressing P450 2C2, and annexin V staining was consistent with the activation of an apoptotic pathway in these cells. Down-regulation of BAP31 with small interfering RNA partially reversed the apoptosis. These results suggest that interaction of P450 2C2 with BAP31 is important for its ER retention and expression level and that BAP31 may be involved in the regulation of apoptosis induced by the ER overload response to increased expression of P450.     

The signal for Golgi retention of bovine beta 1,4-galactosyltransferase is in the transmembrane domain.

The expression and localization of bovine beta 1,4-galactosyltransferase (Gal T) has been studied in mammalian cells transfected with Gal T cDNA constructs, and the role of the amino-terminal domains of Gal T in Golgi localization examined. Here we demonstrate that the transmembrane (signal/anchor) domain of bovine Gal T contains a positive Golgi retention signal. Bovine Gal T was characterized in transfected cells with anti-bovine Gal T antibodies, affinity-purified from a rabbit antiserum using a bacterial recombinant fusion protein. These affinity-purified antibodies recognized native bovine Gal T and showed minimum cross-reactivity with Gal T from non-bovine sources. Bovine Gal T cDNA was expressed, as active enzyme, transiently in COS-1 cells and stably in murine L cells, and the product was shown to be localized to the Golgi complex by immunofluorescence using the polypeptide-specific antibodies. A low level of surface bovine Gal T was also detected in the transfected L cells by flow cytometry. The removal of 18 of the 24 amino acids from the cytoplasmic domain of bovine Gal T did not alter the Golgi localization of the product transiently expressed in COS-1 cells or stably expressed in L cells. Both the full-length bovine Gal T and the cytoplasmic domain deletion mutant were N-glycosylated in the transfected L cells, indicating both proteins have the correct N(in)/C(out) membrane orientation. Deletion of both the cytoplasmic and signal/anchor domains of bovine Gal T and incorporation of a cleavable signal sequence resulted in a truncated soluble bovine Gal T that was rapidly secreted (within 1 h) from transfected COS-1 cells. Replacement of the signal/anchor domain of bovine Gal T with the signal/anchor domain of the human transferrin receptor resulted in the transport of the hybrid molecule to the cell surface of transfected COS-1 cells. Furthermore, a hybrid construct containing the signal/anchor domain of Gal T with ovalbumin was efficiently retained in the Golgi complex, whereas ovalbumin anchored to the membrane by the transferrin receptor signal/anchor was expressed at the cell surface of transfected COS-1 cells. Overall, these studies show that the hydrophobic, signal/anchor domain of Gal T is both necessary and sufficient for Golgi localization.     

调节TGF-β1/Smad信号通路对内质网应激状态下肝癌HepG2细胞凋亡的影响

目的: 探讨TGF-β1/Smad信号通路对内质网应激(ERS)状态下肝癌HepG2细胞凋亡的影响机制。方法: 首先建立内质网应激模型:以3 μmol/L的衣霉素(TM)处理人肝癌HepG2细胞株24 h,诱导细胞发生ERS。实验分为6组,每组3个复孔,实验重复3次,6组分别为:Untreated组(未处理组)、TM组(3 μmol/L TM处理组)、TM+NC组(3 μmol/L TM+si-TGF-β1阴性对照组)、TM+si-TGF-β1组(3 μmol/L TM+si-TGF-β1组)、TM+pEX-3组(3 μmol/L TM+质粒对照组)及TM+TGF-β1 pEX-3组(3 μmol/L TM+TGF-β1过表达质粒组),利用脂质体的方法将TGF-β1小干扰RNA(si-TGF-β1)及TGF-β1过表达质粒(TGF-β1 pEX-3)转染入HepG2细胞,转染24 h后,利用RT-qPCR和Western blot检测各组HepG2细胞TGF-β1/Smad信号通路相关因子TGF-β1、p-Smad2表达的情况;CCK-8和流式细胞术分别检测各组HepG2细胞增殖抑制率和凋亡率变化情况。结果: 与Untreated组相比,TM组细胞的TGF-β1及p-Smad2的表达明显降低(P＜0.05);与TM组相比,TM+si-TGF-β1组细胞的TGF-β1及p-Smad2的表达和细胞的增殖抑制率、凋亡率显著降低(P＜0.01),而TM+TGF-β1 pEX-3组细胞的TGF-β1及p-Smad2的表达和细胞增殖抑制率、凋亡率显著升高(P＜0.01)。结论: TGF-β1/Smad信号通路在肝癌HepG2细胞发生ERS后受到抑制,当该通路被激活后,ERS状态下肝癌HepG2细胞的凋亡率显著升高。     

A common Dubin-Johnson syndrome mutation impairs protein maturation and transport activity of MRP2 (ABCC2)

Absence of a functional multidrug resistance protein 2 (MRP2; symbol ABCC2) from the hepatocyte canalicular membrane is the molecular basis of Dubin- Johnson syndrome, an inherited disorder associated with conjugated hyperbilirubinemia in humans. In this work, we analyzed a relatively frequent Dubin-Johnson syndrome mutation that leads to an exchange of two hydrophobic amino acids, isoleucine 1173 to phenylalanine (MRP2I1173F), in a predicted extracellular loop of MRP2. HEK-293 cells stably transfected with MRP2I1173F cDNA synthesized a mutant protein that was mainly core-glycosylated, predominantly retained in the endoplasmic reticulum, and degraded by proteasomes. MRP2I1173F did not mediate ATP-dependent transport of leukotriene C(4) (LTC(4)) into vesicles from plasma membrane and endoplasmic reticulum preparations while normal MRP2 was functionally active. Human HepG2 cells were used to study localization of MRP2I1173F in a polarized cell system. Quantitative analysis showed that GFP-tagged MRP2I1173F was localized to the apical membrane in only 5% of transfected, polarized HepG2 cells compared with 80% for normal MRP2-GFP. Impaired protein maturation followed by proteasomal degradation of inactive MRP2I1173F explain the deficient hepatobiliary elimination observed in this group of Dubin-Johnson syndrome patients.     

Endoplasmic reticulum retention determinants in the transmembrane and linker domains of cytochrome P450 2C1

The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequence-specific requirement for direct retention in the endoplasmic reticulum.     

Different apoptotic effects of wogonin via induction of H(2)O(2) generation and Ca(2+) overload in malignant hepatoma and normal hepatic cells

Wogonin, a major active constituent of Scutellaria baicalensis, possesses potent anticancer activities both in vivo and in vitro. This paper describes the different apoptotic effects of wogonin in HepG2 and L02 cells and the possible mechanism for the differences. Through DAPI staining, Annexin-V/PI double-staining assay, JC-1 detection and the expressions of the key apoptotic proteins, we find that wogonin prefers to induce apoptosis in HepG2 cells through the mitochondrial pathway, while has much less effects on L02 cells. Moreover, overexpression of Bcl-2 can block wogonin-induced apoptosis in HepG2 cells. To illustrate the specific selective mechanism of wogonin in apoptosis induction, H(2)O(2), (·)O(2)(-) and Ca(2+) are measured by 2',7'-dichlorfluorescein-diacetate, dihydroethidium and Flou-3 AM assay, respectively. The results show that the different apoptotic effects of wogonin in HepG2 and L02 cells are due to the different regulations to the redox balance of reactive oxygen species and the Ca(2+) release from endoplasmic reticulum. IP(3)R-sensitive Ca(2+) channels are the key targets of the wogonin-increased H(2)O(2). Besides, the activation of PLCγ1 plays as a bridge between H(2)O(2) signal molecules and Ca(2+) release. Taken together, wogonin preferentially kills hepatoma cells by H(2)O(2)-dependent apoptosis triggered by Ca(2+) overload. The results reveal that wogonin is a competitive anticancer drug candidate for the malignant hepatoma therapy.     

Disruption of disulfide bonds is responsible for impaired secretion in human complement factor H deficiency

Factor H, a secretory glycoprotein composed of 20 short consensus repeat modules, is an inhibitor of the complement system. Previous studies of inherited factor H deficiency revealed single amino acid substitutions at conserved cysteine residues, on one allele arginine for cysteine 518 (C518R) and on the other tyrosine for cysteine 941 (C941Y) (Ault, B. H., Schmidt, B. Z., Fowler, N. L., Kashtan, C. E., Ahmed, A. E., Vogt, B. A., and Colten, H. R. (1997) J. Biol. Chem. 272, 25168-25175). To ascertain if the phenotype, impaired secretion of factor H, is due to the C518R substitution or the C941Y substitution and to ascertain the mechanism by which secretion is impaired, we studied COS-1 and HepG2 cells transfected with wild type and several mutant factor H molecules. The results showed markedly impaired secretion of both C518R and C941Y factor H as well as that of factor H molecules bearing alanine or arginine substitutions at the Cys518-Cys546 disulfide bond (C518A, C546A, C546R, C518A-C546A). In each case, mutant factor H was retained in the endoplasmic reticulum and degraded relatively slowly as compared with most other mutant secretory and membrane proteins that are retained in the endoplasmic reticulum. These data indicate that impaired secretion of the naturally occurring C518R and C941Y mutant factor H proteins is due to disruption of framework-specific disulfide bonds in factor H short consensus repeat modules.     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Mechanism of endoplasmic reticulum retention of mutant vasopressin precursor caused by a signal peptide truncation associated with diabetes insipidus.

Autosomal dominant neurohypophyseal diabetes insipidus is caused by mutations in the gene encoding the vasopressin precursor protein, prepro-vasopressin-neurophysin II. We analyzed the molecular consequences of a mutation (DeltaG227) recently identified in a Swiss kindred that destroys the translation initiation codon. In COS-7 cells transfected with the mutant cDNA, translation was found to initiate at an alternative ATG, producing a truncated signal sequence that was functional for targeting and translocation but was not cleaved by signal peptidase. The mutant precursor was completely retained within the endoplasmic reticulum. The uncleaved signal did not affect folding of the neurophysin portion of the precursor, as determined by its protease resistance. However, formation of disulfide-linked aggregates indicated that it interfered with the formation of the disulfide bond in vasopressin, most likely by blocking its insertion into the hormone binding site of neurophysin. Preventing disulfide formation in the vasopressin nonapeptide by mutation of cysteine 6 to serine was shown to be sufficient to cause aggregation and retention. These results indicate that the DeltaG227 mutation induces translation of a truncated signal sequence that cannot be cleaved but prevents correct folding and oxidation of vasopressin, thereby causing precursor aggregation and retention in the endoplasmic reticulum.     

The COOH terminus of several liver carboxylesterases targets these enzymes to the lumen of the endoplasmic reticulum

To investigate the potential role of the COOH-terminal peptides in retaining a family of soluble carboxylesterases in the lumen of the endoplasmic reticulum, the pI 6.1 esterase cDNA has been cloned into the pKCR3 vector for transient expression in COS cells. The plasmid-encoded product appeared to be identical to the authentic enzyme: it was active on alpha-naphthyl acetate and behaved as a homotrimer of noncovalently bound 60-kDa subunits which contain a single, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide chain. This enzyme was retained in the transgenic COS cells. In contrast, a mutated form ending in HVER-COOH was secreted, indicating that the natural terminus HVEL-COOH contains topogenic information, with the ultimate Leu residue as an essential part. Variants of pI 6.1 esterase ending in HIEL-COOH, or HTEL-COOH were retained in cells to the same extent as the wild-type protein. Therefore, the sequences HIEL and HTEL present at the COOH termini of several liver esterases (rabbit forms 1 and 2, human esterase, mouse egasyn, and rat pI 6.4 esterase) most likely have a function in their localization in the endoplasmic reticulum. Finally, an HDEL-COOH variant of pI 6.1 esterase was also normally retained, demonstrating that this signal can be correctly decoded by the sorting machinery of mammalian cells. Cell retention signals of the type HXEL-COOH appear to be common in higher eukaryotes and tolerate considerable variation at the antepenultimate X residue.     

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.     

ER Reorganization is Remarkably Induced in COS-7 Cells Accumulating Transmembrane Protein Receptors Not Competent for Export from the Endoplasmic Reticulum

The newly synthesized mutant L501fsX533 Frizzled-4 form and the alpha3beta4 nicotinic acetylcholine receptor expressed in the absence of nicotine accumulate in the endoplasmic reticulum of COS-7 cells and induce the formation of large areas of smooth and highly convoluted cisternae. This results in a generalized block of the transport to the Golgi complex of newly synthesized proteins. Intriguingly, both effects happen peculiarly in COS-7 cells; HeLa, Huh-7, and HEK293 cells expressing the two receptors at similar level than COS-7 cells show normal ER and normal transport toward the plasma membrane. These results question the conclusion that a dominant-negative mechanism would explain the dominance of the mutant L501fsX533 Fz4 allele in the transmission of a form of Familial exudative vitreoretinopathy. Moreover, they indicate that the coordination of endoplasmic reticulum homeostasis in COS-7 cells is particularly error prone. This finding suggests that COS-7 cells may be extremely useful to study the molecular mechanisms regulating endoplasmic reticulum size and architecture.