Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells.

The spontaneously immortalised DF-1 cell line is rapidly replacing its progenitor primary chicken embryo fibroblasts (CEFs) for studies on avian viruses such as avian influenza but no comprehensive study has as yet been reported comparing their innate immunity phenotypes. We conducted microarray analyses of DF-1 and CEFs, under both normal and stimulated conditions using chicken interferon-α (chIFN-α) and the attenuated infectious bursal disease virus vaccine strain PBG98. We found that DF-1 have an attenuated innate response compared to CEFs. Basal expression levels of Suppressor of Cytokine Signalling 1 (chSOCS1), a negative regulator of cytokine signalling in mammals, are 16-fold higher in DF-1 than in CEFs. The chSOCS1 "SOCS box" domain (which in mammals, interacts with an E3 ubiquitin ligase complex) is not essential for the inhibition of cytokine-induced JAK/STAT signalling activation in DF-1. Overexpression of SOCS1 in chIFN-α-stimulated DF-1 led to a relative decrease in expression of interferon-stimulated genes (ISGs; MX1 and IFIT5) and increased viral yield in response to PBG98 infection. Conversely, knockdown of SOCS1 enhanced induction of ISGs and reduced viral yield in chIFN-α-stimulated DF-1. Consequently, SOCS1 reduces induction of the IFN signalling pathway in chicken cells and can potentiate virus replication.

Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.

SREBPs exit the ER in a complex with SCAP. Together, they move to the Golgi where SREBP is cleaved, releasing a fragment that activates genes encoding lipid biosynthetic enzymes. Sterols block ER exit, preventing cleavage, decreasing transcription, and achieving feedback control of lipid synthesis. Here, we report an in vitro system to measure incorporation of SCAP into ER vesicles. When membranes were isolated from sterol-depleted cells, SCAP entered vesicles in a reaction requiring nucleoside triphosphates and cytosol. SCAP budding was diminished in membranes from sterol-treated cells. Kinetics of induction of budding in vitro matched kinetics of ER exit in living cells expressing GFP-SCAP. These data localize the sterol-regulated step to budding of SCAP from ER and provide a system for biochemical dissection.

Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi.

The proteolytic cleavage of sterol regulatory element-binding proteins (SREBPs) is regulated by SREBP cleavage-activating protein (SCAP), which forms complexes with SREBPs in membranes of the endoplasmic reticulum (ER). In sterol-depleted cells, SCAP facilitates cleavage of SREBPs by Site-1 protease, thereby initiating release of active NH(2)-terminal fragments from the ER membrane so that they can enter the nucleus and activate gene expression. In sterol-overloaded cells, the activity of SCAP is blocked, SREBPs remain bound to membranes, and transcription of sterol-regulated genes declines. Here, we provide evidence that sterols act by inhibiting the cycling of SCAP between the ER and Golgi. We use glycosidases, glycosidase inhibitors, and a glycosylation-defective mutant cell line to demonstrate that the N-linked carbohydrates of SCAP are modified by Golgi enzymes in sterol-depleted cells. After modification, SCAP returns to the ER, as indicated by experiments that show that the Golgi-modified forms of SCAP cofractionate with ER membranes on density gradients. In sterol-overloaded cells, the Golgi modifications of SCAP do not occur, apparently because SCAP fails to leave the ER. Golgi modifications of SCAP are restored when sterol-overloaded cells are treated with brefeldin A, which causes Golgi enzymes to translocate to the ER. These studies suggest that sterols regulate the cleavage of SREBPs by modulating the ability of SCAP to transport SREBPs to a post-ER compartment that houses active Site-1 protease.

Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi.

Cholesterol homeostasis in animal cells is achieved by regulated cleavage of membrane-bound transcription factors, designated SREBPs. Proteolytic release of the active domains of SREBPs from membranes requires a sterol-sensing protein, SCAP, which forms a complex with SREBPs. In sterol-depleted cells, SCAP escorts SREBPs from ER to Golgi, where SREBPs are cleaved by Site-1 protease (S1P). Sterols block this transport and abolish cleavage. Relocating active S1P from Golgi to ER by treating cells with brefeldin A or by fusing the ER retention signal KDEL to S1P obviates the SCAP requirement and renders cleavage insensitive to sterols. Transport-dependent proteolysis may be a common mechanism to regulate the processing of membrane proteins.

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N.

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

Topology of SREBP cleavage-activating protein, a polytopic membrane protein with a sterol-sensing domain.

The NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) are released from endoplasmic reticulum membranes by proteases whose activities depend upon SREBP cleavage-activating protein (SCAP), a polytopic endoplasmic reticulum membrane protein. The activity of SCAP is inhibited by sterols, which appear to interact with the polytopic membrane domain of SCAP. Here, we use protease protection and N-linked glycosylation site-mapping techniques to define the topology of the eight membrane-spanning domains of SCAP. The data indicate that the NH2 terminus and COOH terminus of SCAP face the cytosol. The long intralumenal loops after membrane-spanning segments 1 and 7 are glycosylated, confirming their lumenal location. The region comprising membrane-spanning segments 2-6 shows sequence resemblance to putative sterol-sensing domains in three other proteins: 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), the Niemann-Pick C1 protein, and the morphogen receptor Patched. The orientation of the eight membrane-spanning segments in SCAP is consistent with the model proposed for HMG-CoA reductase (Olender, E. H., and Simoni, R. D. (1992) J. Biol. Chem. 267, 4223-4235). The membrane-spanning domains of SCAP and HMG-CoA reductase confer sterol sensitivity upon the functional activities of the two molecules. The common membrane topology of the two proteins is consistent with the notion that sterols regulate both proteins by a common mechanism.

Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence from in vivo competition studies.

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that promote lipid synthesis in animal cells. They are embedded in the membranes of the endoplasmic reticulum (ER) in a helical hairpin orientation and are released from the ER by a two-step proteolytic process. Proteolysis begins when the SREBPs are cleaved at Site-1, which is located at a leucine residue in the middle of the hydrophobic loop in the lumen of the ER. Sterols suppress Site-1 cleavage, apparently by interacting with a polytopic membrane protein designated SREBP cleavage-activating protein (SCAP). SREBPs and SCAP are joined together in ER membranes through interaction of their cytoplasmic COOH-terminal domains. Here we use an in vivo competition assay in transfected cells to show that the SREBP.SCAP complex is essential for Site-1 cleavage. Overexpression of the truncated COOH-terminal domains of either SREBP-2 or SCAP disrupted the complex between full-length SREBP-2 and SCAP as measured by co-immunoprecipitation. This resulted in a complete inhibition of Site-1 cleavage that was restored by concomitant overexpression of full-length SCAP. The transfected COOH-terminal domains also inhibited the transcription of a reporter gene driven by an SRE-containing promoter, and this, too, was restored by overexpression of full-length SCAP. We interpret these data to indicate that the SREBP.SCAP complex directs the Site-1 protease to its target in the lumenal domain of SREBP and that disruption of this complex inactivates the Site-1 cleavage reaction.

Variable aberrant cDNAs in single diphtheria toxin-resistant human fibroblasts.

We treated transformed human fibroblasts with diphtheria toxin (DT) and isolated 40 single cells that were toxin resistant but unable to propagate. In 13 of them toxin resistance was associated with the presence of one or more aberrant transcripts of the structural gene for elongation factor 2 (EF-2). cDNA obtained from these transcripts had 164-447 bp-long deletions. Each of these deletions was associated with 2-8 base pairs-long repeats at its breakpoints. Only 10 out of 16 cDNA deletions were associated with presumed exon junctions. A role is suggested for errors in transcription in producing the aberrant transcripts which gave rise to the deletion-bearing cDNA species.

Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein.

SREBP cleavage-activating protein (SCAP) stimulates the proteolytic cleavage of membrane-bound SREBPs, thereby initiating the release of NH2-terminal fragments from cell membranes. The liberated fragments enter the nucleus and stimulate transcription of genes involved in synthesis and uptake of cholesterol and fatty acids. Sterols repress cleavage of SREBPs, apparently by interacting with the membrane attachment domain of SCAP. In the present studies we show that SCAP, like the SREBPs, is located in membranes of the endoplasmic reticulum and nuclear envelope. The COOH-terminal domain of SCAP, like that of the SREBPs, is located on the cytosolic face of the membranes. Co-immunoprecipitation experiments show that SCAP and SREBP-2 form a complex that can be precipitated with antibodies to either component. Complex formation occurs when cells express only the COOH-terminal domain of either SREBP-2 or SCAP, indicating that the complex forms between the two COOH-terminal domains. Truncation of SREBP-2 at its COOH terminus prevents the formation of complexes with SCAP and simultaneously reduces proteolytic cleavage. We conclude that proteolytic cleavage of SREBPs requires the formation of a complex with the COOH-terminal domain of SCAP and that SCAP is therefore a required element in the regulation of sterol and fatty acid metabolism in animal cells.

Molecular analysis of T-lymphocyte HPRT- mutations in individuals exposed to ionizing radiation in Goiânia, Brazil.

We have characterized 54 HPRT- point mutations in T-lymphocytes from 17 individuals exposed to ionizing radiation of 137Cs in Goiania, Brazil and compared this spectrum to that of 30 HPRT- mutants from 9 unexposed Brazilian controls. The average internal exposure of the exposed group was 205 mCi, and the average external exposure was 1.7 Gy. The average HPRT- mutant frequency for the exposed group was 13.3 x 10(-5), approximately a 10-fold increase over the mutant frequency of the unexposed controls, which was 1.56 x 10(-5). The types of point mutations characterized included base substitutions, small deletions, frameshifts, insertions, complex mutations, and losses of exon sequences from the mRNA. The relative frequency of the different mutation types was similar in the two studied groups. However, in our study the distribution of events within the hprt coding sequence seemed to cluster at the same regions of the gene. These observations imply that the hprt gene does not present a homogeneous target to radiation mutagenesis, and perhaps this class of information may be used to detect radiation exposure in human populations.

Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein.

Through expression cloning we have isolated a cDNA-encoding SREBP cleavage-activating protein (SCAP), which regulates cholesterol metabolism by stimulating cleavage of transcription factors SREBP-1 and -2, thereby releasing them from membranes. The cDNA was isolated from Chinese hamster ovary cells with a dominant mutation that renders them resistant to sterol-mediated suppression of cholesterol synthesis and uptake. Sterol resistance was traced to a G-->A transition at codon 443 of SCAP, changing aspartic acid to asparagine. The D443N mutation enhances the cleavage-stimulating ability of SCAP and renders it resistant to inhibition by sterols. SCAP has multiple membrane-spanning regions, five of which resemble the sterol-sensing domain of HMG CoA reductase, an endoplasmic reticulum enzyme whose degradation is accelerated by sterols. SCAP appears to be a central regulator of cholesterol metabolism in animal cells.

Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines.

Oxygenated sterols such as 25-hydroxycholesterol kill Chinese hamster ovary cells because they inhibit the proteolytic processing of sterol regulatory element binding proteins (SREBPs), a pair of membrane-bound transcription factors that activate genes controlling cholesterol synthesis and uptake from lipoproteins. The unprocessed SREBPs remain membrane-bound, they cannot activate the cholesterol biosynthetic pathway, and the cells die of cholesterol deprivation. Several sterol-resistant hamster cell lines have been isolated previously by chemical mutagenesis and selection for resistance to killing by 25-hydroxycholesterol. We recently identified the defect in one such cell line (25-RA cells) as a point mutation in a newly discovered membrane protein of 1276 amino acids, designated SREBP cleavage-activating protein (SCAP). The mutation in the 25-RA cells resulted from a G-to-A transition in codon 443 of the SCAP gene, changing aspartic acid to asparagine. Wild-type SCAP, when overexpressed by transfection, stimulates the proteolytic processing of both SREBPs. The D443N substitution is an activating mutation that increases the activity of SCAP and renders it resistant to inhibition by 25-hydroxycholesterol. We here report the identical G-to-A transition in two additional lines of Chinese hamster ovary cells that were mutagenized and isolated by a similar protocol. The three mutations occurred independently as indicated by haplotype analysis of the mutant genes using two intragenic sequence polymorphisms. All three cell lines were mutagenized with alkylating agents (nitrosoethylurea or ethylmethane sulfonate) that favor G-to-A transitions. Nevertheless, the finding of the same nucleotide substitution at the same location in all three cell lines indicates that SCAP may be unique in its ability to stimulate SREBP cleavage, and residue 443 is a crucial determinant of the protein's ability to be inhibited by 25-hydroxycholesterol.

Increased hprt mutant frequencies in Brazilian children accidentally exposed to ionizing radiation.

We have examined the effects of ionizing radiation on somatic mutations in vivo, using the hprt clonal assay. The study was performed on blood samples obtained from children exposed during a radiological accident that happened in 1987, in Goiânia, Brazil. The group of children exposed to ionizing radiation includes six males and four females ranging in age from 6 to 14 years at the time of exposure. The radiation doses ranged from 15 to 70 cGy. A Brazilian control group, not exposed to ionizing radiation, was also analyzed under similar conditions. the mean hprt mutant frequency for the exposed group was 4.6 times higher than the control group, although the cloning efficiency from the exposed group was significantly reduced. Linear regression analysis of the mutant frequency and ionizing radiation dose did not show a significant relationship between these two parameters. However, a reliable inverse relationship was demonstrated when the regression analysis was performed with nonselective cloning efficiency and ionizing radiation dose. It was demonstrated that nonselective cloning efficiency diminishes as ionizing radiation dose increases. To correct mutant frequencies for clonal events, the clonal relationship between the hprt mutant clones was examined by T-cell receptor analysis. The majority of the mutants analyzed represented individual clones, thus validating the observed mutant frequencies.

Repair of DNA double-strand breaks induced in Saccharomyces cerevisiae using different gamma-ray dose-rates: a pulsed-field gel electrophoresis analysis.

We investigated the effects of gamma-ray exposures at high dose-rate (HDR, 23.2 Gy/min) and low dose-rate (LDR, 0.47 Gy/min) on survival and the induction of DNA double-strand breaks (dsb) in a diploid wild-type (D7) and the repair-deficient mutant strain rad52/rad52 of Saccharomyces cerevisiae. Analysis by pulsed-field gel electrophoresis (PFGE) using a contour homogeneous electric field apparatus revealed that, at HDR, in the range 0-400 Gy, dsb are induced as a linear function of gamma-ray dose. Liquid holding recovery in non-nutrient medium (LHR) for 48 h of wild-type cells treated at HDR, significantly increased survival and reduced the yield of dsb. Such changes did not occur in rad52/rad52 cells defective in the repair of dsb. Thus, in gamma-irradiated wild-type cells, an efficient repair of dsb is taking place during LHR. Treatments of wild-type cells at LDR resulted in higher survival and an approximately two-fold lower yield of dsb than at HDR. Such a dose-rate effect was absent in rad52/rad52 cells suggesting that, in wild-type cells during LDR exposures, significant amounts of dsb can be repaired. This repair could be very much accentuated by 48-h LHR of wild-type cells treated at LDR. The relationship observed between gamma-ray survival and dsb repair clearly indicates that increases in survival of wild-type cells, during LDR as compared with HDR exposures and after LHR, are strongly related to the repair of dsb.