Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.     

ClusFCM: an algorithm for predicting protein functions using homologies and protein interactions

We introduce a new algorithm, called ClusFCM, which combines techniques of clustering and fuzzy cognitive maps (FCM) for prediction of protein functions. ClusFCM takes advantage of protein homologies and protein interaction network topology to improve low recall predictions associated with existing prediction methods. ClusFCM exploits the fact that proteins of known function tend to cluster together and deduce functions not only through their direct interaction with other proteins, but also from other proteins in the network. We use ClusFCM to annotate protein functions for Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worm), and Drosophila melanogaster (fly) using protein-protein interaction data from the General Repository for Interaction Datasets (GRID) database and functional labels from Gene Ontology (GO) terms. The algorithm's performance is compared with four state-of-the-art methods for function prediction--Majority, chi(2) statistics, Markov random field (MRF), and FunctionalFlow--using measures of Matthews correlation coefficient, harmonic mean, and area under the receiver operating characteristic (ROC) curves. The results indicate that ClusFCM predicts protein functions with high recall while not lowering precision. Supplementary information is available at http://www.egr.vcu.edu/cs/dmb/ClusFCM/.     

Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence

The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3'-untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression

The nucleotide and amino acid sequences for rat type I angiotensin II receptor were deduced through molecular cloning and sequence analysis of its complementary DNAs. The rat angiotensin II receptor consists of 359 amino acid residues and has a sequence similar to G protein-coupled receptors. The expression of this receptor gene was detected in the adrenal, liver and kidney by Northern blotting. Sodium deprivation positively modulated the expression of the receptor gene in the adrenal. No detectable change was observed in the expression levels of this receptor gene between spontaneously hypertensive rats and Wistar-Kyoto rats in the tissues examined including the adrenal, brain, kidney and liver. Interestingly the expression of this receptor gene was developmentally regulated.     

Examination of protein sequence homologies: IV. Twenty-seven bacterial ferredoxins

Summary Sequence homologies of 27 bacterial ferredoxins were examined using a computer program that quantitatively evaluates extent of similarity as a correlation coefficient. The results of a similarity search among the sequences demonstrated that the basal sequence consists of a pair of extremely similar segments of 26 amino acids connected by a three-amino acid group. The segment pairs, which would have arisen from gene duplication, are termed the first and second units. Because of the gene duplication, the connector sequence appears to have been introduced as a structurally important chain reversal. Each of the two units contains four cysteine residues, which are inserted one by one among seven, two, two, three, and eight amino acid alignments, respectively. The bacterial ferredoxins were categorized with regard to basal constitution as follows: group 1, in which both units closely conform to the basal structure; group 2, in which the second unit is modified in a characteristic manner among members; group 3, in which the first unit is modified in a characteristic manner, while the conforming second unit is accompanied by a long accessory sequence; group 4, in which there are modifications before and/or after the units, of which the respective central domains remain nearly intact; and group 5, where only the former of two Fe:S cluster ligation sets of four cysteines is estimated to remain intact, whereas the latter set is extremely modified. It is noteworthy that throughout all bacterial ferredosins, one of two cysteine sets never fails to be completely intact and, moreover, the connector of three amino acids also exists intact. Based on this grouping and on the correspondences among the groups, average correlation coefficients among all members were computed, and the respective evolutionary relationships were examined. The results supported the proposition that transposition had occurred in theAzotobacter-type ferredoxins of group 3.     

Gene transfer, expression, and molecular cloning of the human transferrin receptor gene

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk- cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5' end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3' untranslated sequences that are not required for receptor expression in L cells.     

Intracellular interactions of transferrin and its receptor during biosynthesis

The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfide-linked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and is ultimately delivered to an acidic compartment, where Tf releases its Fe3+. We did not observe conversion of Tf to apoTf in the TGR, showing that the part of the TGR passed by secreted Tf has a pH higher than 5.5. We conclude that when a ligand-receptor combination is synthesized by one and the same cell, ligand and receptor can interact during biosynthesis and be transported to the cell surface.     

cDNA cloning of the murine transferrin receptor: sequence of trans-membrane and adjacent regions

We previously purified the murine transferrin receptor from cultured myeloma cells and determined the amino acid sequence of six tryptic peptides. We now report the cloning of cDNA encoding the murine transferrin receptor. A tryptic peptide containing a region of six consecutive amino acids, all encoded by four or fewer codons, was used to design two overlapping oligonucleotide probes, 17 and 14 nucleotides in length. These probes were used to screen a lambda gt10 cDNA library from NS-1 myeloma cells. Of approximately 400,000 plaques screened, two hybridized strongly to both probes. A subfragment of one clone that hybridized with both oligonucleotide probes was found to encode the tryptic peptide from which the probes were derived, as well as another sequenced tryptic peptide. Comparison of the sequence with that of the human transferrin receptor shows a high degree of conservation of the sequences surrounding and penetrating the membrane, including cysteine residues that may be involved in interchain disulfide bonding and/or covalent attachment of lipid. The current data, when combined with the published sequence of the human receptor, allow assignment of all six tryptic peptides to a single chain, supporting the idea that the receptor is a homodimer. A 4.9-kb messenger RNA was found in several cultured murine and human tumor cell lines, but transferrin receptor messenger RNA was not detectable in murine spleen. An additional RNA species of 2.7 kb was present in approximately equal abundance in the murine myelomas NS-1 and C118 but was absent from T lymphomas TIKAUT, ST-1, and ST-4.     

Chicken transferrin receptor gene: conservation 3'' noncoding sequences and expression in erythroid cells.

Recombinant clones of the chicken transferrin receptor gene and cDNA have been isolated and sequenced. Two highly conserved regions have been identified in the 3' noncoding sequence of the human and chicken TR gene. The conserved regions include sequences that have been shown to be involved in the iron-dependent regulation of human TR mRNA stability. These sequences can be modeled as two different types of RNA secondary structures, one containing stem-loop structures that are similar to the iron-responsive elements found in ferritin mRNA and the other being a stable, duplex/stem-loop structure. Both forms show considerable similarity between chicken and human mRNA. The expression of TR is developmentally regulated during erythroid maturation, and immature erythroid cells express exceptionally high levels of TR mRNA.     

Cloning and expression of the Haemophilus influenzae transferrin receptor genes

The genomic transferrin receptor genes ( tbpA and tbpB  ) from two strains of Haemophilus influenzae type b (Hib) and two strains of non-typable H. influenzae (NTHi) have been cloned and sequenced. The deduced protein sequences of the H. influenzae tbpA genes were 95–100% conserved and those of the tbpB genes were 66–100% conserved. The tbpB gene from one strain of NTHi was found to encode a truncated Tbp2 protein. The tbpB genes from four additional NTHi strains were amplified by the polymerase chain reaction (PCR) utilizing primers derived from the conserved N-terminal sequences of Tbp1 and Tbp2 and were found to encode full-length proteins. Although several bacterial species express transferrin receptors, when the Tbp1 and Tbp2 sequences from different organisms were compared, there was only limited homology. Recombinant Tbp1 and Tbp2 proteins were expressed from Escherichia coli and antisera were raised to the purified proteins. There was significant antigenic conservation of both Tbp1 and Tbp2 amongst H. influenzae strains, as determined by Western blot analysis. In a passive model of bacteraemia, infant rats were protected from challenge with Hib after transfer of anti-rTbp2 antiserum, but not after anti-rTbp1 antiserum.     

Isolation and sequence analysis of promoter DNA fragments of the Luciferin-binding protein gene inGonyaulax polyedra

To understand the unusual features of the genes and genomes fromGonyaulax polyedra, we isolated the promoter portions of the luciferin binding protein (LBP) gene, using IPCR methods, and characterized their sequences. Five LBP genomic clones were classified into a group of genes from the LBPα family, based on the sequence homology of the coding portion of the LBP gene. They were subdivided into two groups. Southern analysis implied that the promoter region is conserved well in most LBP genes. The comparison of the promoter regions from the LBP and luciferase genes showed that, although some portions of their sequences were well conserved, these two genes did not share common features of promoter region, as is normally found in eukaryotes or prokaryotes.     

Cloning, expression and sequence homologies of cDNA for human gamma enolase

The nucleotide sequence of the human gamma-enolase mRNA was determined from recombinant cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5'-noncoding region of 74 bp and a 897-bp-long 3'-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat gamma-enolase. Both the 5'- and 3'-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat gamma-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of gamma-enolase mRNAs. The size of the gamma-enolase mRNA expressed in human brain is 2.4 kb. A crosshybridizing 1.5-kb message is detected in human skeletal muscle which may be derived from the beta-enolase-coding gene.     

Hemopexin-dependent down-regulation of expression of the human transferrin receptor

To investigate the regulation mechanism of the uptake of iron and heme iron by the cells and intracellular utilization of iron, we examined the interaction between iron uptake from transferrin and hemopexin-mediated uptake of heme by human leukemic U937 cells or HeLa cells. U937 cells exhibited about 40,000 hemopexin receptors/cell with a dissociation constant (Kd) of 1 nM. Heme bound in hemopexin was taken up by U937 cells or HeLa cells in a receptor-mediated manner. Treatment of both species of cells with hemopexin led to a rapid decrease in iron uptake from transferrin in a hemopexin dose-dependent manner, and the decrease seen in case of treatment with hemin was less than that seen with hemopexin. The decrease of iron uptake by hemopexin contributed to a decrease in cell surface transferrin receptors on hemopexin-treated cells. Immunoblot analysis of the transferrin receptors revealed that the cellular level of receptors in U937 cells did not vary during an 8-h incubation with hemopexin although the number of surface receptors as well as iron uptake decreased within the 2-h incubation. After 4 h of incubation of the cells with hemopexin, a decrease of the synthesis of the receptors occurred. Thus, the down-regulation of transferrin receptors by hemopexin can be attributed to at least two mechanisms. One is a rapid redistribution of the surface receptor into the interior of the cells, and the other is a decrease in the biosynthesis of the receptor. 59Fe from the internalized heme rapidly appeared in non-heme iron (ferritin) coincidently with the induction of heme oxygenase. The results suggest that iron released from heme down-regulates the expression of the transferrin receptors and iron uptake.     

Frequent promoter hypermethylation and expression reduction of the glucocorticoid receptor gene in breast tumors

Previous studies have found that expression of the Glucocorticoid Receptor (GR) is altered or reduced in various cancers, while the GR promoter has been shown to be methylated in gastric, lung, and colorectal cancers. Examining a small cohort of matched normal and breast cancer samples we found that GR levels were dramatically reduced in almost all tumors in relation to their normal tissue. The methylation status of the GR promoter was assessed to determine if this observed decrease of expression in breast tumors could be due to epigenetic regulation. While it was not methylated in normal tissue, the GR proximal promoter was methylated in 15% of tumor samples, particularly, but not exclusively, in Estrogen Receptor positive tumors. GR expression in these tumors was particularly low and loss of GR expression was specifically correlated with methylation of the proximal promoter GR B region. Overall, these results show that hypermethylation of the promoter in tumors is a frequent event and suggests that GR may act as a tumor suppressor in breast tissue.     

Frequent promoter hypermethylation and expression reduction of the glucocorticoid receptor gene in breast tumors

Previous studies have found that expression of the Glucocorticoid Receptor (GR) is altered or reduced in various cancers, while the GR promoter has been shown to be methylated in gastric, lung, and colorectal cancers. Examining a small cohort of matched normal and breast cancer samples we found that GR levels were dramatically reduced in almost all tumors in relation to their normal tissue. The methylation status of the GR promoter was assessed to determine if this observed decrease of expression in breast tumors could be due to epigenetic regulation. While it was not methylated in normal tissue, the GR proximal promoter was methylated in 15% of tumor samples, particularly, but not exclusively, in Estrogen Receptor positive tumors. GR expression in these tumors was particularly low and loss of GR expression was specifically correlated with methylation of the proximal promoter GR B region. Overall, these results show that hypermethylation of the promoter in tumors is a frequent event and suggests that GR may act as a tumor suppressor in breast tissue.