The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an R... more The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an RNA polymerase sharing characteristics with polymerase I, but there is no sequence homology between them nor between these promoters and ribosomal promoters. We report the detailed characterization of the VSG promoter. The 70-bp region upstream of the transcription start site was sufficient for full promoter activity. Mutational
The American journal of tropical medicine and hygiene, 2002
In the search for new diagnostic methods that would distinguish Trypanosoma brucei rhodesiense fr... more In the search for new diagnostic methods that would distinguish Trypanosoma brucei rhodesiense from T. b. brucei and T. b. gambiense, we have developed two polymerase chain reaction (PCR) primer sets. The first primer set was derived from the serum resistance-associated (SRA) gene of T. b. rhodesiense that confers resistance to lysis by normal human serum (NHS). The specificity of the SRA-based PCR was tested on 97 different trypanosome populations originating from various taxonomic groups, host species, and geographic regions. Only one of 25 T. b. rhodesiense samples was negative in this PCR, and none of 72 other samples were positive in this assay. Interestingly, a reference T. brucei strain (TREU927/4) currently used for genome sequencing was negative for the SRA gene; however, this strain was resistant to lysis by NHS. The second primer set was derived from a specific variant surface glycoprotein (VSG) expression site where the SRA gene is expressed (R-ES). This primer set ident...
African trypanosomes can spend a long time in the blood of their mammalian host, where they are e... more African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?
Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. ... more Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. The basis of this pathology is the resistance of these parasites to lysis by normal human serum (NHS). Resistance to NHS is conferred by a gene that encodes a truncated form of the variant surface glycoprotein termed serum resistance associated protein (SRA). We show that SRA is a lysosomal protein, and that the amino-terminal alpha-helix of SRA is responsible for resistance to NHS. This domain interacts strongly with a carboxy-terminal alpha-helix of the human-specific serum protein apolipoprotein L-I (apoL-I). Depleting NHS of apoL-I, by incubation with SRA or anti-apoL-I, led to the complete loss of trypanolytic activity. Addition of native or recombinant apoL-I either to apoL-I-depleted NHS or to fetal calf serum induced lysis of NHS-sensitive, but not NHS-resistant, trypanosomes. Confocal microscopy demonstrated that apoL-I is taken up through the endocytic pathway into the lysosome...
Serpins (serine protease inhibitors) are a large family of struc- turally related proteins found ... more Serpins (serine protease inhibitors) are a large family of struc- turally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses re- vealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in
We present an overview of the regulation of vsg expression, focusing on initiation and elongation... more We present an overview of the regulation of vsg expression, focusing on initiation and elongation of transcription as well as processing and stabilization of the transcripts. We propose a model where common factors are involved in the reverse controls of the genes for the two main stage-specific antigens, the Vsg and procyclin: a cross-talk between the two transcription units would allow a fast rerouting of limiting factors at differentiation, thereby allowing the expression of only one type of antigen at a time. A similar mechanism would ensure that only one vsg ES is fully expressed at a time in bloodstream forms.
The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune respo... more The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of variant surface glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription-PCR (RT-PCR) to analyse transcription of different segments of the expression sites in different variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.
Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. ... more Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. The basis of this pathology is the resistance of these parasites to lysis by normal human serum (NHS). Resistance to NHS is conferred by a gene that encodes a truncated form of the variant surface glycoprotein termed serum resistance associated protein (SRA). We show that SRA is a lysosomal protein, and that the amino-terminal alpha-helix of SRA is responsible for resistance to NHS. This domain interacts strongly with a carboxy-terminal alpha-helix of the human-specific serum protein apolipoprotein L-I (apoL-I). Depleting NHS of apoL-I, by incubation with SRA or anti-apoL-I, led to the complete loss of trypanolytic activity. Addition of native or recombinant apoL-I either to apoL-I-depleted NHS or to fetal calf serum induced lysis of NHS-sensitive, but not NHS-resistant, trypanosomes. Confocal microscopy demonstrated that apoL-I is taken up through the endocytic pathway into the lysosome. We propose that apoL-I is the trypanosome lytic factor of NHS, and that SRA confers resistance to lysis by interaction with apoL-I in the lysosome.
Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ri... more Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ricinus. It belongs to the superfamily of serpins and interferes with both haemostasis and the immune response of the host. In this study, we first show that Iris is expressed in nymphs but not in the female midgut nor in males. We also show that Iris is present in the saliva. To examine its potency as anti-tick vaccine candidate, we set up three models of I. ricinus infestation on immunized animals: nymphs on mice, and adults and nymphs on rabbits. We report the rise of neutralizing antibodies following immunization of rabbits and mice. This comes with a significant protective immunity against ticks in rabbits only, resulting in a 30% mortality rate and a diminution of weight gain in both nymphs and adults and a prolongation of blood feeding time in adults. This is the first report on an anti-tick vaccine trial on I. ricinus using a protein able to interact with both host immunity and haemostasis, as a vaccinating antigen.
Proceedings of the National Academy of Sciences, 2007
Apolipoprotein L-I (apoL-I) is a human high-density lipoprotein (HDL) component able to kill Tryp... more Apolipoprotein L-I (apoL-I) is a human high-density lipoprotein (HDL) component able to kill Trypanosoma brucei brucei by forming anion-selective pores in the lysosomal membrane of the parasite. Another HDL component, haptoglobin-related protein (Hpr), has been suggested as an additional toxin required for full trypanolytic activity of normal human serum. We recently reported the case of a human lacking apoL-I (apoL-I ؊/؊ HS) as the result of frameshift mutations in both apoL-I alleles. Here, we show that this serum, devoid of any trypanolytic activity, exhibits normal concentrations of HDL-bound Hpr. Conversely, the serum of individuals with normal HDL-bound apoL-I but who lack Hpr and haptoglobin [Hp(r) ؊/؊ HS] as the result of gene deletion (anhaptoglobinemia) exhibited phenotypically normal but delayed trypanolytic activity. The trypanolytic properties of Hp(r) ؊/؊ HS were mimicked by free recombinant apoL-I, whereas recombinant Hpr did not affect trypanosomes. The lysis delay observed with either Hp(r) ؊/؊ HS or recombinant apoL-I could entirely be attributed to a defect in the uptake of the lytic components. Thus, apoL-I is responsible for the trypanolytic activity of normal human serum, whereas Hpr allows fast uptake of the carrier HDL particles, presumably through their binding to an Hp/Hpr surface receptor of the parasite.
Background: During their blood meal, ticks secrete a wide variety of proteins that can interfere ... more Background: During their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.
Background: During their blood meal, ticks secrete a wide variety of proteins that interfere with... more Background: During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.
In Trypanosoma brucei, the mutually exclusive expression of the major surface antigens, the varia... more In Trypanosoma brucei, the mutually exclusive expression of the major surface antigens, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the procyclic form, is due to a stage-specific accumulation of the respective mRNAs. Through the targeting of a reporter construct in the procyclin promoter region, we show that independently of any selection pressure, a relatively high level of transcription (approximately 10%) occurs from the procyclin promoter in the bloodstream form. This transcription leads to the production of detectable amounts of polyadenylated mRNAs. However, RNA elongation in the procyclin transcription unit is down-regulated at this stage. Transcription elongation in the procyclin and VSG units is inversely controlled by the combination of factors which cause the differentiation of bloodstream into procyclic forms in vitro. These factors include temperature, citrate/cis-aconitate and the incubation medium. Our results suggest that inverse regulations of primary transcription in the VSG and procyclin units are early events that underly the differentiation of the parasite.
| African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasi... more | African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasites that infect a wide range of mammals. Human blood, unlike the blood of other mammals, has efficient trypanolytic activity, and this needs to be counteracted by these parasites. Resistance to this activity has arisen in two subspecies of Trypanosoma brucei -Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense -allowing these parasites to infect humans, and this results in sleeping sickness in East Africa and West Africa, respectively. Study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of an ionic-pore-forming apolipoprotein -known as apolipoprotein L1 -that is associated with high-density-lipoprotein particles in human blood. In this Opinion article, we argue that apolipoprotein L1 is the factor that is responsible for the trypanolytic activity of human serum.
The results demonstrated that the mono-allelic control allowing preferential RNA production from ... more The results demonstrated that the mono-allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady-state ES transcripts, particularly the VSG mRNA , were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run-on transcription analysis indicated that RNA polymerase was still fully associated with the promoter-proximal half of the 'active' ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the 'active' ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ . This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.
The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its pr... more The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.
The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an R... more The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an RNA polymerase sharing characteristics with polymerase I, but there is no sequence homology between them nor between these promoters and ribosomal promoters. We report the detailed characterization of the VSG promoter. The 70-bp region upstream of the transcription start site was sufficient for full promoter activity. Mutational
The American journal of tropical medicine and hygiene, 2002
In the search for new diagnostic methods that would distinguish Trypanosoma brucei rhodesiense fr... more In the search for new diagnostic methods that would distinguish Trypanosoma brucei rhodesiense from T. b. brucei and T. b. gambiense, we have developed two polymerase chain reaction (PCR) primer sets. The first primer set was derived from the serum resistance-associated (SRA) gene of T. b. rhodesiense that confers resistance to lysis by normal human serum (NHS). The specificity of the SRA-based PCR was tested on 97 different trypanosome populations originating from various taxonomic groups, host species, and geographic regions. Only one of 25 T. b. rhodesiense samples was negative in this PCR, and none of 72 other samples were positive in this assay. Interestingly, a reference T. brucei strain (TREU927/4) currently used for genome sequencing was negative for the SRA gene; however, this strain was resistant to lysis by NHS. The second primer set was derived from a specific variant surface glycoprotein (VSG) expression site where the SRA gene is expressed (R-ES). This primer set ident...
African trypanosomes can spend a long time in the blood of their mammalian host, where they are e... more African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?
Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. ... more Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. The basis of this pathology is the resistance of these parasites to lysis by normal human serum (NHS). Resistance to NHS is conferred by a gene that encodes a truncated form of the variant surface glycoprotein termed serum resistance associated protein (SRA). We show that SRA is a lysosomal protein, and that the amino-terminal alpha-helix of SRA is responsible for resistance to NHS. This domain interacts strongly with a carboxy-terminal alpha-helix of the human-specific serum protein apolipoprotein L-I (apoL-I). Depleting NHS of apoL-I, by incubation with SRA or anti-apoL-I, led to the complete loss of trypanolytic activity. Addition of native or recombinant apoL-I either to apoL-I-depleted NHS or to fetal calf serum induced lysis of NHS-sensitive, but not NHS-resistant, trypanosomes. Confocal microscopy demonstrated that apoL-I is taken up through the endocytic pathway into the lysosome...
Serpins (serine protease inhibitors) are a large family of struc- turally related proteins found ... more Serpins (serine protease inhibitors) are a large family of struc- turally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses re- vealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in
We present an overview of the regulation of vsg expression, focusing on initiation and elongation... more We present an overview of the regulation of vsg expression, focusing on initiation and elongation of transcription as well as processing and stabilization of the transcripts. We propose a model where common factors are involved in the reverse controls of the genes for the two main stage-specific antigens, the Vsg and procyclin: a cross-talk between the two transcription units would allow a fast rerouting of limiting factors at differentiation, thereby allowing the expression of only one type of antigen at a time. A similar mechanism would ensure that only one vsg ES is fully expressed at a time in bloodstream forms.
The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune respo... more The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of variant surface glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription-PCR (RT-PCR) to analyse transcription of different segments of the expression sites in different variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.
Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. ... more Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. The basis of this pathology is the resistance of these parasites to lysis by normal human serum (NHS). Resistance to NHS is conferred by a gene that encodes a truncated form of the variant surface glycoprotein termed serum resistance associated protein (SRA). We show that SRA is a lysosomal protein, and that the amino-terminal alpha-helix of SRA is responsible for resistance to NHS. This domain interacts strongly with a carboxy-terminal alpha-helix of the human-specific serum protein apolipoprotein L-I (apoL-I). Depleting NHS of apoL-I, by incubation with SRA or anti-apoL-I, led to the complete loss of trypanolytic activity. Addition of native or recombinant apoL-I either to apoL-I-depleted NHS or to fetal calf serum induced lysis of NHS-sensitive, but not NHS-resistant, trypanosomes. Confocal microscopy demonstrated that apoL-I is taken up through the endocytic pathway into the lysosome. We propose that apoL-I is the trypanosome lytic factor of NHS, and that SRA confers resistance to lysis by interaction with apoL-I in the lysosome.
Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ri... more Iris is a specific elastase inhibitor expressed in the salivary glands of the hard tick Ixodes ricinus. It belongs to the superfamily of serpins and interferes with both haemostasis and the immune response of the host. In this study, we first show that Iris is expressed in nymphs but not in the female midgut nor in males. We also show that Iris is present in the saliva. To examine its potency as anti-tick vaccine candidate, we set up three models of I. ricinus infestation on immunized animals: nymphs on mice, and adults and nymphs on rabbits. We report the rise of neutralizing antibodies following immunization of rabbits and mice. This comes with a significant protective immunity against ticks in rabbits only, resulting in a 30% mortality rate and a diminution of weight gain in both nymphs and adults and a prolongation of blood feeding time in adults. This is the first report on an anti-tick vaccine trial on I. ricinus using a protein able to interact with both host immunity and haemostasis, as a vaccinating antigen.
Proceedings of the National Academy of Sciences, 2007
Apolipoprotein L-I (apoL-I) is a human high-density lipoprotein (HDL) component able to kill Tryp... more Apolipoprotein L-I (apoL-I) is a human high-density lipoprotein (HDL) component able to kill Trypanosoma brucei brucei by forming anion-selective pores in the lysosomal membrane of the parasite. Another HDL component, haptoglobin-related protein (Hpr), has been suggested as an additional toxin required for full trypanolytic activity of normal human serum. We recently reported the case of a human lacking apoL-I (apoL-I ؊/؊ HS) as the result of frameshift mutations in both apoL-I alleles. Here, we show that this serum, devoid of any trypanolytic activity, exhibits normal concentrations of HDL-bound Hpr. Conversely, the serum of individuals with normal HDL-bound apoL-I but who lack Hpr and haptoglobin [Hp(r) ؊/؊ HS] as the result of gene deletion (anhaptoglobinemia) exhibited phenotypically normal but delayed trypanolytic activity. The trypanolytic properties of Hp(r) ؊/؊ HS were mimicked by free recombinant apoL-I, whereas recombinant Hpr did not affect trypanosomes. The lysis delay observed with either Hp(r) ؊/؊ HS or recombinant apoL-I could entirely be attributed to a defect in the uptake of the lytic components. Thus, apoL-I is responsible for the trypanolytic activity of normal human serum, whereas Hpr allows fast uptake of the carrier HDL particles, presumably through their binding to an Hp/Hpr surface receptor of the parasite.
Background: During their blood meal, ticks secrete a wide variety of proteins that can interfere ... more Background: During their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.
Background: During their blood meal, ticks secrete a wide variety of proteins that interfere with... more Background: During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.
In Trypanosoma brucei, the mutually exclusive expression of the major surface antigens, the varia... more In Trypanosoma brucei, the mutually exclusive expression of the major surface antigens, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the procyclic form, is due to a stage-specific accumulation of the respective mRNAs. Through the targeting of a reporter construct in the procyclin promoter region, we show that independently of any selection pressure, a relatively high level of transcription (approximately 10%) occurs from the procyclin promoter in the bloodstream form. This transcription leads to the production of detectable amounts of polyadenylated mRNAs. However, RNA elongation in the procyclin transcription unit is down-regulated at this stage. Transcription elongation in the procyclin and VSG units is inversely controlled by the combination of factors which cause the differentiation of bloodstream into procyclic forms in vitro. These factors include temperature, citrate/cis-aconitate and the incubation medium. Our results suggest that inverse regulations of primary transcription in the VSG and procyclin units are early events that underly the differentiation of the parasite.
| African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasi... more | African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasites that infect a wide range of mammals. Human blood, unlike the blood of other mammals, has efficient trypanolytic activity, and this needs to be counteracted by these parasites. Resistance to this activity has arisen in two subspecies of Trypanosoma brucei -Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense -allowing these parasites to infect humans, and this results in sleeping sickness in East Africa and West Africa, respectively. Study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of an ionic-pore-forming apolipoprotein -known as apolipoprotein L1 -that is associated with high-density-lipoprotein particles in human blood. In this Opinion article, we argue that apolipoprotein L1 is the factor that is responsible for the trypanolytic activity of human serum.
The results demonstrated that the mono-allelic control allowing preferential RNA production from ... more The results demonstrated that the mono-allelic control allowing preferential RNA production from a given ES stops during this process. Accordingly, the steady-state ES transcripts, particularly the VSG mRNA , were strongly reduced. However, transcripts from the beginning of different ESs were still synthesized, and in vitro run-on transcription analysis indicated that RNA polymerase was still fully associated with the promoter-proximal half of the 'active' ES. Analysis of transcripts from two central tandem genes confirmed the existence of a residual decreasing transcriptional gradient in the 'active' ES of SS forms. Thus, in these forms the RNA polymerase of the ES is inactivated in situ . This inactivation is accompanied by a strong overall reduction of nuclear DNA transcription. Although cAMP is involved in the LS to SS transformation, no direct effect of cAMP was observed on the VSG ES control.
The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its pr... more The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.
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Papers by Luc Vanhamme