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PubMed1997

Regulated processing of hepatitis C virus core protein is linked to subcellular localization.

Posttranslational processing and subcellular localization of the HCV core protein are critical steps involved in the assembly of hepatitis C virus (HCV). In this study, both of these events were investigated by in vitro translation and transient COS-1 cell transfection of core protein expression constructs. Mutations at amino acid residues 173 to 174 and 191 to 192 disrupted processing events at the two putative cleavage sites in the C-terminal hydrophobic region of the core protein, indicating that these residues are implicated in the pathway of core protein maturation. As a result, two forms of core protein, C173 and C191, were detected by immunoblotting. Indirect immunofluorescence experiments showed that core proteins C173 and C191, when produced from HCV full-length protein or various polyprotein precursors, displayed a cytoplasmic localization. The C173 species, however, was translocated to the nucleus when expressed in the absence of C191. These findings indicate that preferential cleavage may occur during core protein maturation and that the association of the C191 with the C173 species may contribute to the distinct subcellular distribution of core protein. This may provide a possible mechanism for the control of the diverse biological functions of core protein during HCV replication and assembly.

Amino Acid Sequence↗
PubMed1997

The apolipoprotein E and beta-fibrinogen G/A-455 gene polymorphisms are associated with ischemic stroke involving large-vessel disease.

The relationship between the apolipoprotein E (apoE) and beta-fibrinogen G/A-455 polymorphisms and cerebrovascular disease (CVD) was examined in the present study. We compared 227 patients with the subtypes of CVD (large-vessel disease, lacunar stroke, cardiac embolism, or undetermined pathomechanisms) with 225 control subjects. The occurrence of apoE isoforms (E2, E3, and E4) and the beta-fibrinogen G/A-455 genotype was determined in these individuals. No differences in apoE polymorphisms or allele frequencies between the CVD patients and control subjects were found. However, analysis of apoE genotypes as a function of stroke subtype revealed that the apoE4 allele was significantly more common in those patients with macroangiopathy-associated CVD. The only CVD risk factor that distinguished patients with the E4 allele from those with other apoE genotypes was elevated cholesterol. No association between the beta-fibrinogen G/A-455 polymorphism and CVD was found. However, homozygosity for the A allele was more common in patients with CVD resulting from large-vessel disease. These data demonstrate that the apoE4 allele and the AA genotype of the beta-fibrinogen G/A-455 polymorphism occur significantly more frequently in patients with CVD resulting from stenosis of large, brain-supplying vessels. Such genetic analyses may further our understanding of the etiology of cerebrovascular disease.

Adult↗
PubMed2002

Estrogen receptor alpha and activating protein-1 mediate estrogen responsiveness of the progesterone receptor gene in MCF-7 breast cancer cells.

The progesterone receptor (PR) gene is activated by estrogen in MCF-7 human breast cancer cells. Although the human PR gene does not contain an estrogen response element (ERE), we have identified a putative activating protein-1 (AP-1) site at +90 in the PR gene that was hypersensitive to deoxyribonuclease I cleavage in genomic Southern analysis, bound purified Fos and Jun, formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts in gel mobility shift assays, and functioned as an estrogen-responsive enhancer in transient cotransfection assays. When the +90 AP-1 site was mutated in the context of the PR gene, estrogen responsiveness was significantly decreased. Purified estrogen receptor (ER) enhanced binding of Fos and Jun to the +90 AP-1 site and bound to an adjacent imperfect ERE half-site. Mutating this ERE half-site diminished the binding of ER, Fos, and Jun and decreased transcription. Chromatin immunoprecipitation assays demonstrated that the ER, Fos, and Jun were present at the +90 AP-1 site in the endogenous PR gene only after treatment of MCF-7 cells with estrogen. These studies suggest that the cooperative interaction of the ER with Fos and Jun proteins helps confer estrogen responsiveness to the endogenous PR gene.

Binding Sites↗
PubMed2000

Requirement for C-terminal end of fibroblast growth factor receptor 4 in translocation of acidic fibroblast growth factor to cytosol and nucleus.

The ability of COS cells to bind and internalise acidic fibroblast growth factor (aFGF) was studied after transient transfection of the cells with wild-type and mutated fibroblast growth factor receptor 4. In one case the tyrosine kinase of the receptor was inactivated by a point mutation in the active site, whereas in other cases parts of the receptor were deleted to remove various parts of the cytoplasmic domain. In all cases the receptors were expressed at the cell surface at a high level and the cells bound labelled growth factor efficiently and internalised it by endocytosis. Translocation of externally added aFGF across cellular membranes to reach the cytosol and nucleus was measured as transport of labelled growth factor to the nuclear fraction obtained by centrifugation, by farnesylation of growth factor modified to carry a CAAX motif, and by phosphorylation of the growth factor at a site specific for protein kinase C. Whereas both full-length receptors (with and without an active kinase domain) facilitated translocation of the growth factor to the cytosol and nucleus, as assessed by these methods, the mutants of the receptor where the C terminus was deleted, were unable to do so. In contrast, a receptor containing only the 57 most C-terminal amino acids of the cytoplasmic domain in addition to the juxtamembrane, transmembrane and extracellular domains, was in fact able to mediate translocation of aFGF to the cytosol. These data indicate that information contained in the C terminus of the receptor is required for translocation.

Animals↗
PubMedDate not supplied

Proto-oncogene N-myc promoter is down regulated by the Wilms' tumor suppressor gene WT1.

The Wilms' tumor 1 (WT1) gene is a tumor suppressor gene that encodes a zinc-finger transcription factor. WT1 represses transcription of several growth factors and growth factor receptors. The N-myc proto-oncogene encodes a transcription factor which regulates cell growth and differentiation. N-myc is coexpressed with WT1 in the developing kidney and is overexpressed in many Wilms' tumors. Here, we show that the proto-oncogene N-myc promoter was down-regulated by WT1 in transient transfection assays. However, mutant WT1 (R394W) which has a mutation in the DNA binding domain could not repress the N-myc promoter. Electrophoretic mobility shift assays showed that the oligonucleotides containing the WT1 motifs could bind recombinant WT1 proteins. This suggests that the repression of the N-myc promoter is mediated through the WT1 binding sites. This finding may help to elucidate the relationship of WT1 and N-myc in tumorigenesis and renal development.

Binding Sites↗
PubMed1991

Epidermal growth factor and other mitogens induce binding of a protein complex to the c-fos serum response element in human astrocytoma and other cells.

Rapid stimulation of c-fos transcription by many agonists requires the serum response element (SRE), which binds at least two distinct nuclear proteins, p67SRF and p62TCF. Using nuclear protein extracts from 1321-N1 human astrocytoma cells, we investigated ligand-induced changes in binding of these proteins to SRE probes. In these cells c-fos mRNA expression can be induced by epidermal growth factor (EGF) through protein kinase C-independent pathways and by phorbol esters through protein kinase C. We detected two DNA-protein complexes that formed specifically with the SRE (bands 1 and 2). Band 2 formation was increased 4-6 min after stimulation with EGF as well as serum and phorbol esters; this peaked at 10-30 min and returned to basal levels by 60 min. Induction of band 2 formation preceded the onset and peak accumulation of c-fos mRNA (15 and 30 min after EGF stimulation, respectively) and its return to basal levels (by 1-2 h). Band 2 formation was also increased A431 cells stimulated with EGF and in HeLa and Swiss-3T3 cells stimulated with serum. We found that band 1 contained p67SRF bound to the SRE; band 2 contained p67SRF and a second protein. Gel shift analyses using [35S]methionine-labeled p67SRF and nonradioactive DNA probes suggested that hormone treatment most likely modified the second protein component of band 2. Transient transfection of 1321-N1 cells with plasmids containing point mutations that prevented band 2 formation in vitro also abolished induction of c-fos transcription in vivo as assayed by RNase protection analysis. Thus, hormone-stimulated formation of the protein-DNA complex represented by band 2 may be involved in the activation of c-fos transcription.

Astrocytoma↗
PubMed1993

Thyroid hormone receptor dimerization is required for dominant negative inhibition by mutations that cause thyroid hormone resistance.

The syndrome of thyroid hormone resistance (THR) is caused by multiple distinct mutations of the ligand-binding domain of the thyroid hormone beta receptor. Although the mutant receptors are transcriptionally inactive, they inhibit normal receptor function in a dominant negative manner to cause hormone resistance. Because most of the naturally occurring mutations are clustered within two areas that lie on either side of a putative dimerization region, we hypothesized that receptor dimerization was important for dominant negative inhibition. In gel mobility shift assays, two THR mutants (G345R and P453H) formed homodimers as well as heterodimers with the retinoic acid X receptor alpha. In contrast, an artificial mutation (L428R) in one of the hydrophobic heptad repeats of the putative receptor dimerization domain impaired heterodimerization with retoinoic acid X receptor alpha without altering the formation of homodimers. Double mutants containing either of the THR mutations along with the dimerization mutation formed homodimers but not heterodimers, reflecting the properties of the dimerization mutant alone. In transient expression assays using positively (TRETKLuc) or negatively (TSH alpha Luc) regulated reporter genes, the dominant negative activity of the THR mutants was eliminated by the addition of the dimerization mutation. These results support a mechanism for dominant negative activity by THR mutants in which functionally inactive heterodimers bind to DNA to inhibit access by normal receptors.

Amino Acid Sequence↗
PubMed1996

Critical role of Ras in the proliferation and prevention of apoptosis mediated by IL-2.

Activation of the Ras small guanine nucleotide-binding proteins upon IL-2 stimulation of T lymphocytes has been described previously. However, the relevance of the Ras molecules in the cellular events triggered by IL-2 still remained to be clarified. To analyze the effects of Ras inhibition in early proliferative responses to IL-2, we have transiently expressed a Ras dominant negative mutant carrying a point mutation in codon 17 (Ser17Asn or N17) under a tetracycline-controlled expression system. We have found that Ras has a crucial role in both proliferation and prevention of apoptosis through IL-2R, while IL-4 promotes proliferation and inhibits apoptosis by Ras-independent signals. The use of flow cytometry has allowed us to correlate the effects in proliferation and apoptosis with the expression level of the Ras mutant among the transfected population. Furthermore, the availability of a murine T cell line that can be maintained independently in the presence of IL-2 or IL-4 has provided specificity controls, since IL-4 does not trigger Ras activation.

Animals↗
PubMed1996

Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences.

Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF. Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs). To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region. Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR. We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs. Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect. The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation. Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

Animals↗
PubMed2006

Unusual clinical presentation and possible rescue of a novel claudin-16 mutation.

CONTEXT: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is caused by a dysfunction of Claudin-16 (CLDN16) and characterized by renal wasting of Mg(2+) and Ca(2+). OBJECTIVE: The objectives of this study were to study the clinical parameters in suspected FHHNC patients, identify mutations in the CLDN16 gene, and analyze molecular defects associated with the mutant protein. DESIGN, SETTING, AND PARTICIPANTS: CLDN16 genes from two siblings diagnosed with FHHNC were sequenced. Expression and characterization of the mutant protein in renal MDCK cells were studied. OUTCOME MEASURES: Standard urine and serum parameters to diagnose FHHNC were determined. Mutations in the CLDN16 gene were identified. The subcellular distribution of the mutant protein was analyzed by immunofluorescence microscopy. RESULTS: Urine and blood analysis showed signs typical for FHHNC. One patient, in addition, presented with hypocalcemic tetany, a phenomenon so far not described for FHHNC. Both siblings carry a novel mutation in CLDN16, Y207X. The review of medical records showed that hypocalcemia is not uncommon in the early childhood of FHHNC patients. Expressed in MDCK cells, the Y207X mutant is not detected at tight junctions but instead is found in lysosomes and, to a lesser extent, the endoplasmic reticulum. Surface expression can be rescued by inhibiting clathrin-mediated internalization. CONCLUSIONS: We propose that mutations in CLDN16 are considered in childhood hypocalcemia. CLDN16 Y207X is transiently delivered to the plasma membrane but not retained and is rapidly retrieved by internalization. Inhibitors of endocytosis may provide novel therapeutic strategies.

Amino Acid Sequence↗
PubMed1992

Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II.

Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disease of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), a 454-amino acid protein encoded by a gene with 12 exons. To identify the causative mutations in five TAT alleles cloned from three RHS patients, chimeric genes constructed from normal and mutant TAT alleles were tested in directing TAT activity in a transient expression assay. DNA sequence analysis of the regions identified as nonfunctional revealed six different point mutations. Three RHS alleles have nonsense mutations at codons 57, 223, and 417, respectively. One "complex" RHS allele carries a GT----GG splice donor mutation in intron 8 together with a Gly----Val substitution at amino acid 362. A new splice acceptor site in intron 2 of the fifth RHS allele leads to a shift in reading frame.

Adolescent↗
PubMed1997

HPA-10w(b) (La(a)): genetic determination of a new platelet-specific alloantigen on glycoprotein IIIa and its expression in COS-7 cells.

The heterodimeric complex glycoprotein (GP)IIb-IIIa, the fibrinogen receptor of platelets, carries numerous alloantigen systems. These polymorphisms are responsible for the immune response after transfusion or during pregnancy. In the latter case, the mother develops an antibody against an epitope present on fetal platelets, and this results in platelet destruction in the fetus. In this report, we describe the molecular characterization of a new alloantigen (La(a)) on GPIIIa responsible for neonatal alloimmune thrombocytopenia (NAIT). Using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and DNA sequencing, we found a point mutation (G to A) in a heterozygous state on the GPIIIa gene leading to amino acid substitution Arg to Gln at position 62 of the mature protein. Transient expression of GPIIb-IIIa complexes in Cos-7 cells using wild-type or mutated GPIIIa cDNA allowed us to demonstrate that this mutation was responsible for expression of the La(a) epitope.

Adult↗
PubMed2004

E1B-55-kilodalton protein is not required to block p53-induced transcription during adenovirus infection.

The adenovirus E1B-55-kDa protein binds and inactivates the tumor suppressor protein p53. However, the role of this interaction during infection is still poorly understood and was therefore examined here. Infection with a virus carrying the E1B-55-kDa mutation R239A, preventing the interaction with p53, led to the accumulation of p53. However, p53 target genes were not activated in the infected cells, although p53 phosphorylation did occur and the p53 antagonists Mdm2 and deltaNp73 did not accumulate. Deletion of E4orf6, alone or in combination with E1B-55-kDa, did not allow the induction of p53-responsive genes either. In transient reporter assays, the viral E1A-13S protein antagonized p53 activity; mutational analysis suggested that this depends partially on p300 binding, but it depends even more strongly on the interaction of E1A with the p400/TRRAP protein complex. However, viruses expressing E1A mutants lacking these binding activities, in combination with E1B-55-kDa R239A, still abolished p53 activity. In contrast, when the mutation of E1B-55-kDa at R239A was combined with a deletion of the apoptosis inhibitor E1B-19-kDa, infected cells showed more extensive apoptosis than after infection with single mutants, suggesting that accumulated p53, albeit transcriptionally inactive, might nonetheless enhance apoptosis. Despite extensive apoptosis of the infected cells, the deletion of E1B-19-kDa, in combination with the E1B-55-kDa mutation or in the presence of the constitutively active p53 mutant p53mt24-28, reduced virus replication less than fivefold. In conclusion, adenovirus does not need direct binding of E1B-55-kDa to inactivate p53, and forced p53 activity with consecutive apoptosis does not severely impair virus replication.

Adenovirus E1A Proteins↗
PubMed1999

Human papillomavirus type 31 oncoproteins E6 and E7 are required for the maintenance of episomes during the viral life cycle in normal human keratinocytes.

The E6 and E7 oncoproteins of the high-risk human papillomavirus (HPV) types are able to immortalize human keratinocytes in vitro and likely contribute to the development of anogenital malignancies in vivo. The role of these oncoproteins in the productive viral life cycle, however, is not known. To begin to examine these possible roles, mutations in E6 were introduced in the context of the complete HPV 31 genome. Although transfected wild-type HPV 31 genomes, as well as genomes containing an E6 translation termination linker, an E6 frameshift mutation, and a point mutation in the p53 interacting domain were able to replicate in transient assays, only the wild-type genome was stably maintained as an episome. Interestingly, mutant genomes in either the E6 splice-donor site or splice-acceptor site were reduced in replication ability in transient assays; however, cotransfection of E1 and E2 expression vectors restored this function. In a similar fashion, genomes containing mutant HPV 31 E7 genes, including a translation termination mutant, two Rb-binding site mutants, a casein kinase II phosphorylation site mutant, and a transformation deficient mutant, were constructed. Although transient replication was similar to wild type in all of the E7 mutants, only the casein kinase II mutant had the ability to maintain high copies of episomal genomes. These findings suggest a role for E6 and E7 in the viral life cycle beyond their ability to extend the life span of infected cells.

Casein Kinase II↗
PubMed2005

An intermediate form of juvenile Paget's disease caused by a truncating TNFRSF11B mutation.

Juvenile Paget's disease (JPD) is a rare condition with an autosomal recessive mode of inheritance. Typically presenting in infancy or early childhood, the disorder is characterized by a generalized widening of the long bones and thickening of the skull combined with sustained elevation of serum alkaline phosphatase levels. The extremely rapid bone turnover results in osteopenia, fractures, and progressive skeletal deformity. In 2002, mutations in TNFRSF11B, the gene encoding osteoprotegerin, were described as underlying JPD. We evaluated a patient with JPD at the clinical, biochemical, radiological, and molecular level. Mutation analysis of TNFRSF11B revealed a homozygous insertion/deletion in exon 5, predicted to result in truncation of the protein at amino acid 325. The residual activity of the mutated protein product was investigated by Western blotting and ELISA upon transient overexpression. Absence of the C-terminal domain abolished homodimerization and was shown to lead to a decreased capacity of the mutant protein to bind its ligand RANKL. We conclude that truncation of the C-terminal part of osteoprotegerin negatively affects functional activity. As a consequence, osteoclast formation and function are up-regulated, causing the increased bone turnover seen in this patient.

Adult↗
PubMed1989

Some mutations affecting neural or muscular tissues alter the physiological components of the electroretinogram in Drosophila.

Mutants displaying generalized behavioral defects and one mutant having an enzyme deficiency were examined for electroretinogram (ERG) defects. Mutations in nine genes were examined that cause ERG defects. Two, parats4 and slrpD, cause reversibly temperature dependent loss of the off-transients in the ERG. stnC and Tyr-2 cause loss of the on and off-transients. The transient defect in Tyr-2 mapped close to a site shown to affect tyrosinase activity in this strain. Mutations bas, rex and sesD delay recovery from the prolonged depolarization afterpotential. The visual defects of mutations elavjl and nbAEE171 are not complemented by lethal mutations, which, presumably, affect other tissues.

Animals↗
PubMed1991

Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat.

We have compared the relative importance of transcription regulatory regions in the U3 and R regions of the human immunodeficiency virus type 1 long terminal repeat (LTR) by using linker-scanning mutational analysis. Twenty-six mutant LTR-chloramphenicol acetyltransferase (CAT) transient expression plasmids were prepared in which consecutive 18-bp regions of wild-type LTR were replaced with an NdeI-XhoI-SalI (NXS) polylinker. The mutant LTR-CAT plasmids were transfected into unstimulated Jurkat cells, Jurkat cells stimulated with phytohemagglutinin and tetradecanoylphorbol acetate, and Jurkat cells which constitutively express the human immunodeficiency virus type 1 trans-activator protein, Tat. Transcriptional activity was measured by analysis of CAT activity. The activities of these mutants identified one major and several minor transcription control elements in addition to previously identified elements. In addition, this fine-structure analysis identified differences in utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells. A significant regulatory region was indicated by linker-scanning mutations between nucleotides -183 and -130 (relative to the transcription start site, +1). These mutations caused marked decreases in activity of the LTR in unstimulated and especially in stimulated Jurkat cells but had no effect in Tat-expressing Jurkat cells. DNA mobility shift studies comparing probes of wild-type and mutant sequences in the -183 to -130 region indicated that alterations in specific DNA binding correspond to the altered transcriptional activity of the mutants. The effects of mutations in several regulatory regions, in addition to the -183 to -130 region described above, differ between Tat-expressing and -nonexpressing Jurkat cells. For example, the NF-kB sites are necessary for transcription in both Tat-expressing and -nonexpressing cells. However, Tat-expressing Jurkat cells primarily require only the 3'-proximal site, while both stimulated and unstimulated Jurkat cells appear to require both sites. Mutants downstream of the TATA element cause a more significant decrease in activity in Tat-expressing Jurkat cells than in the others. Finally, several mutations in the 5' half of the LTR (-453 to -184) show modest increases in transcription (1.5-fold or less) in unstimulated Jurkat cells only, suggesting possible negative regulatory sites. In summary, our studies have identified a control region (-183 to -130) upstream of the NF-kB sites and have more precisely defined significant differences in the utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells.

Base Sequence↗
PubMed1991

The upstream region of the human gamma-globin gene promoter. Identification and functional analysis of nuclear protein binding sites.

The promoter of the human gamma-globin gene confers tissue specificity as well as developmental stage specificity to gamma gene expression. Earlier work in our laboratory suggested that a fragment of the gamma-globin promoter between -300 and -137 base pairs upstream of the transcription start site contributed to the developmental specificity of the promoter. In this paper, we have mapped potential regulatory elements within this upstream region of the gamma promoter by a combination of in vitro DNA-protein binding assays and functional determinations of promoter strength in transient expression studies. Four sites between -300 and -130 bind proteins present in nuclear extracts of erythroid and non-erythroid cell lines. Mutation of these binding sites by internal base substitution determined that three of the four influence overall promoter strength in transient assays. We have focused on two protein binding sites, -246 to -212 and -195 to -170, that have been reported to bind erythroid-specific factors. The erythroid binding protein NF-E1 and a ubiquitous octamer protein footprint the -195 to -170 site. While internal mutation of this site did not significantly alter promoter strength, a point mutation at position -175 that is associated with hereditary persistence of fetal hemoglobin increased the activity of a promoter construct 20-fold in erythroid cells. A detailed mutational analysis of this site suggests that NF-E1 binding is necessary but not sufficient for activation of the promoter by the -175 mutation, and we propose that a second protein or co-activator is required. The nucleotides between -246 and -212 appear to bind a complex of at least three proteins, at the core of which is a protein binding to a string of dA:dT residues. This complex also appears to form on the 3' A gamma-globin enhancer, and homologous sites have been identified within the locus activating region of the beta-globin cluster, suggesting that this element may mediate long range interactions with distant regulatory elements.

Base Sequence↗