Biomedical subjects

G Tell

Publications and source records attributed to G Tell.

At least 19 recordsLinked to original sources

PubMed1999

Calreticulin enhances the transcriptional activity of thyroid transcription factor-1 by binding to its homeodomain.

Transcription factors are often regulated by associated protein cofactors that are able to modify their activity by several different mechanisms. In this study we show that calreticulin, a Ca2+-binding protein with chaperone activity, binds to thyroid transcription factor-1 (TTF-1), a homeodomain-containing protein implicated in the differentiation of lung and thyroid. The interaction between calreticulin and TTF-1 appears to have functional significance because it results in increased transcriptional stimulation of TTF-1-dependent promoters. Calreticulin binds to the TTF-1 homeodomain and promotes its folding, suggesting that the mechanism involved in stimulation of transcriptional activity is an increase of the steady-state concentration of active TTF-1 protein in the cell. We also demonstrate that calreticulin mRNA levels in thyroid cells are under strict control by the thyroid-stimulating hormone, thus implicating calreticulin in the modulation of thyroid gene expression by thyroid-stimulating hormone.

Binding Sites↗

PubMed1999

Co-operation between the PAI and RED subdomains of Pax-8 in the interaction with the thyroglobulin promoter.

Pax proteins are transcription factors that play an important role in the differentiation of several cell types. These proteins bind to specific DNA sequences through the paired domain. This evolutionarily conserved element is composed of two subdomains (PAI and RED), located at the N- and C-terminals, respectively. Due to the presence of these two subdomains, Pax proteins may recognize DNA in different modes, a possibility that has not been exhaustively explored yet. The C site of the thyroglobulin promoter is bound by the thyroid-specific transcription factor Pax-8. In this study we have characterized the mode by which the Pax-8 paired domain interacts with the C site. Results allow the identification of the respective positions of the PAI and RED subdomains when the full-length protein is bound to the C site. The binding of the isolated PAI and RED subdomains to the C site and to several related mutants was also evaluated. Both subdomains interact with DNA as a monomer and display a lower binding affinity than the full-length protein. Therefore, the Pax-8 paired domain-C site interaction occurs through a co-operation between the two subdomains. The binding properties of the PAI subdomain suggest that the co-operation between PAI and RED subdomains does not merely consist of the sum of contacts established by the single subdomain: the presence of the RED subdomain is necessary for correct DNA recognition by the PAI subdomain, thus accounting for a sort of chronology of events during DNA binding. Since the RED subdomain is much more variable than the PAI subdomain among Pax proteins, these results could explain how distinct Pax proteins may select different target genes.

Base Sequence↗

PubMed1999

Topology of the thyroid transcription factor 1 homeodomain-DNA complex.

The topology of the thyroid transcription factor 1 homeodomain (TTF-1HD)-DNA complex was investigated by a strategy which combines limited proteolysis and selective chemical modification experiments with mass spectrometry methodologies. When limited proteolysis digestions were carried out with the protein in the absence or presence of its target oligonucleotide, differential peptide maps were obtained from which the amino acid residues involved in the interaction could be inferred. Similarly, selective acetylation of lysine residues in both the isolated and the complexed homeodomain allowed us to identify the amino acids protected by the interaction with DNA. Surface topology analysis of isolated TTF-1HD performed at neutral pH was in good agreement with the three-dimensional structure of the molecule as determined by NMR studies under acidic conditions. Minor differences were detected in the C-terminal region of the protein which, contrary to NMR data, showed no accessibility to proteases. Analysis of the complex provided an experimental validation of the model proposed on the basis of the homology with the homeodomain structures described so far. An increased accessibility of the C-terminal region was observed following the interaction, suggesting its displacement from the protein core by the oligonucleotide molecule. Comparative experiments with DNA fragments differing in sequence and binding capabilities highlighted structural differences among the complexes, mainly located in the N-terminal region of the homeodomain, thus accounting for their different dissociation constants.

Acetylation↗

PubMed1998

Ref-1 controls pax-8 DNA-binding activity.

Redox potential controls the DNA-binding activity of several transcription factors. In some cases, the regulation of DNA-binding activity by the redox state is mediated by the Ref-1 nuclear protein. In this study, we demonstrate that Ref-1 is able to induce "in vitro" the DNA-binding activity of the Pax-8 paired domain. In co-transfection experiments, Ref-1 increases the Pax-8 activating effect on thyroglobulin promoter. Moreover, immunoreactivity data suggest that, in nuclear extracts of thyroid cells, the levels of Ref-1 correlate with the amounts of reduced Pax-8. Therefore, the regulation of the Pax-8 DNA-binding activity by redox potential, that we have demonstrated occurring "in vitro", could represent a means to control "in vivo" the function of Pax proteins. Alignment of the Paired domains sequences present in the Protein Data Bank demonstrates a strong conservation of Cys residues, suggesting that the redox regulation of the Paired domain DNA-binding activity is widely conserved along phylogenesis.

Amino Acid Sequence↗

PubMed1998

Redox potential controls the structure and DNA binding activity of the paired domain.

Pax proteins are transcriptional regulators controlling a variety of cell fates during animal development. This role depends on the intact function of the paired (Prd) domain that is able to recognize specific DNA sequences. The Prd domain is composed of two distinct helix-turn-helix subdomains, PAI and RED. Molecular functions of Pax proteins are subjected to different levels of regulation involving both pre-translational and post-translational mechanisms. By using Pax-5 and Pax-8 recombinant proteins, we demonstrate that the binding activity of the Prd domain is regulated through the oxidation/reduction of conserved cysteine residues. Mass spectrometry analysis and mutagenesis experiments demonstrate that the redox regulation is accomplished through the reversible formation of an intramolecular disulfide bridge involving the cysteines present in the PAI subdomain, whereas the RED subdomain appears quite insensitive to redox potential. Circular dichroism experiments indicate that only the reduced form of the Prd domain is able to undergo the proper conformational change necessary for sequence-specific DNA binding. Nuclear extracts from different cell lines contain an activity that is able to reduce the Paired domain and, therefore, to control the DNA binding activity of this protein. Immunodepletion of nuclear extracts demonstrate that the protein Ref-1 contributes to the redox regulation of the Prd DNA binding activity. Given the modular nature of the Prd domain and the independent DNA binding specificity of the PAI and RED subdomains, we propose that this control mechanism should be involved in "switching" among different DNA sequences and therefore different target genes.

Animals↗

PubMed1998

Fibronectin binding promotes a PKC-dependent modulation of NF-kappa B in human T cells.

NF-kappa B was identified as one of the transcription factors leading to antigen-independent stimulation through activation of integrin receptors. This effect was dependent upon stimulation of alpha 4 beta 1 and alpha 5 beta 1 integrins, the major fibronectin-binding integrins of Jurkat T cells, since either RGD or CS-1 peptides at 10(-4) M could prevent NF-kappa B activation. At variance with fibroblasts and smooth muscle cells, in which only p50 and p65 components of the NF-kappa B complex are induced, adhesion of T cells to fibronectin resulted in a strong upregulation of p50 and c-Rel and in a partial increase in p65 activity. The upregulation of NF-kappa B activity was abrogated by calphostin C, an inhibitor of protein kinase C. Cell adhesion determined a strong reduction in the cytoplasmic levels of the NF-kappa B inhibitor I kappa B alpha, reduction that was prevented after treatment with calphostin C, suggesting that PKC-dependent I kappa B alpha phosphorylation might be involved in the upregulation of NF-kappa B.

Binding, Competitive↗

PubMed1998

Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains.

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

Amino Acid Sequence↗

PubMed1998

Pax-8 protein levels regulate thyroglobulin gene expression.

Pax proteins are transcription factors that control differentiation of several cell types. In adult organisms Pax-8 is expressed in the follicular thyroid cell where it interacts with sequences of thyroglobulin and thyroperoxidase promoters. In this study, we provide evidence indicating that Pax-8 protein levels regulate thyroglobulin gene transcription. The most critical approach consisted in increasing Pax-8 protein levels by transfecting thyroid cells with a Pax-8 expression vector. In this situation the thyroglobulin promoter transcriptional activity was significantly increased with respect to untransfected cells. In contrast, the transfection of thyroid transcription factor-1 (TTF-1) expression vector causes a modest decrease of thyroglobulin promoter activity, rather than an increase. Northern blots of human papillary cancers reveal a significant correlation between Pax-8 and thyroglobulin mRNAs. Gel-retardation assays suggest that the mechanism by which the Pax-8 protein levels modulate thyroglobulin promoter activity may occur through competition with TTF-1 for a common binding site. Since we also demonstrate that Pax-8 expression is subjected to TSH control, our data strongly suggest that Pax-8 protein levels could represent an important determinant for the regulation of thyroid cells.

Base Sequence↗

PubMed1997

High mobility group I proteins interfere with the homeodomains binding to DNA.

Homeodomains (HDs) constitute the DNA binding domain of several transcription factors that control cell differentiation and development in a wide variety of organisms. Most HDs recognize sequences that contain a 5'-TAAT-3' core motif. However, the DNA binding specificity of HD-containing proteins does not solely determine their biological effects, and other molecular mechanisms should be responsible for their ultimate functional activity. Interference by other factors in the HD/DNA interaction could be one of the processes by which HD-containing proteins achieve the functional complexity required for their effects on the expression of target genes. Using gel-retardation assay, we demonstrate that two members of the high mobility group I (HMGI) family of nuclear proteins (HMGI-C and HMGY) can bind to a subset of HD target sequences and inhibit HDs from binding to the same sequences. The inhibition of the HD/DNA interaction occurs while incubating HMGI-C with DNA either before or after the addition of the HD. The reduced half-life of the HD.DNA complex in the presence of HMGI-C, and the shift observed in the CD spectra recorded upon HMGI-C binding to DNA, strongly suggest that structural modifications of the DNA are responsible for the inhibition of the HD.DNA complex formation. Moreover, by co-transfection experiments we provide evidence that this inhibition can occur also in vivo. The data reported here would suggest that HMGI proteins may be potential regulators of the function of HD-containing proteins and that they are able to interfere with the access of the HD to their target genes.

3T3 Cells↗

PubMed1997

Functional interference between contacting amino acids of homeodomains.

In a protein, the function of an amino acid at some position depends on the amino acids at other positions. Here we demonstrate a functional interference between base-contacting amino acids (at positions 50 and 54) of homeodomains. When, in the context of Antennapedia or Goosecoid homeodomains, Lys50 is paired to Tyr54 or Ala54 and Gln50 is paired to Met54, the resulting proteins efficiently discriminate among different DNA sequences. In contrast, in the presence of the pair Lys50-Met54, both homeodomains show a reduced capability to discriminate among different DNA sequences. Sequence selection experiments performed in the context of the Goosecoid homeodomain suggest that the presence of Met54 precludes the base-discriminating function of Lys50. These results may explain why the pair Lys50-Met54 is never found in natural homeodomains.

Amino Acid Sequence↗

PubMed1997

Biphasic control of NF-kappa B activation induced by the triggering of HLA-DR antigens expressed on B cells.

The regulation of NF-kappa B activation following the triggering of HLA-DR antigens by mAb L243 has been studied at various times in Raji cells. Electrophoretic mobility shift assays demonstrated a strong increase of NF-kappa B DNA binding after triggering of HLA-DR antigens. Using TNF-alpha-activity neutralizing antibodies, the authors demonstrated that the upregulation of NF-kappa B was found to depend, at later time point, on an autocrine effect of TNF-alpha secreted following triggering of HLA-DR antigens. In contrast, it was found to be TNF-alpha independent in the early time point. Moreover, the upregulation of NF-kappa B binding activity is regulated by the triggering of selected epitopes of HLA-DR antigens. In fact, mAb L243 but not the staphylococcal superantigens, staphylococcal exotoxin toxic shock syndrome toxin-I or staphylococcal enterotoxin B, regulate the NF-kappa B binding activity.

Antibodies, Monoclonal↗

PubMed1997

Hydrogen-deuterium exchange studies of the rat thyroid transcription factor 1 homeodomain.

The 1H NMR solution structure of the rat thyroid transcription factor 1 homeodomain (TTF-1 HD) showed that the molecule folds like classical homeodomains. The C-terminal extension of helix III (fragment 51-59) appeared to adopt a helical geometry, albeit not as rigid as the preceding portion, but the hydrogen-deuterium exchange of backbone amides and the NOE data provided evidence of a discontinuity between the two moieties of helix III at the highly conserved fragment Asn51-His52-Arg53. Analysis of quantitative measurements of isotope exchange rates allows one to recognize the general occurrence, in that region of HD motifs, of opposite effects to helix III stability. Asparagine, histidine and arginine residues occur most frequently at the beginning and end of protein helices. In TTF-1 HD a local fluctuation is observed in the fragment 51-53 which either kinks or tightens the alpha-helix. A search through the protein structure database reveals that the three most common variants of HD fragments 51-53 are often involved in helices and, frequently, in helix initiation or termination. For homeodomains in general, the nature of the fragment 51-53 may be related to the conformational dynamics of their DNA-recognition helix (helix III). Besides the specific results on fragment 51-53, the complete isotope exchange analysis of TTF-1 HD data shows that the partially solvent-exposed recognition helix is stabilized by hydrophobic interactions, like most of the structured regions of the molecule. Hydrophobic stabilization of the contacting regions meets the requirements of a DNA-interaction mechanism which, as shown with other DNA-protein complexes, should entail negative heat capacity variations due to changes in solvent exposure of the nonpolar protein surface.

Amides↗

PubMed1997

Glyceraldehyde 3-phosphate-induced DNA or protein modifications severely inhibit the protein/DNA interaction.

In this study, the effect of the reducing sugar glyceraldehyde 3-phosphate on protein/DNA interaction has been investigated. Treatment with glyceraldehyde 3-phosphate of oligonucleotides recognized by various transcription factors severely inhibits protein binding. The inhibitory effect is time and dose-dependent. Treatment with glyceraldehyde 3-phosphate of the homeodomain protein TTF-1 HD has also an inhibitory effect on the interaction with DNA, again in a time and dose-dependent manner. These "in vitro" effects could have "in vivo" counterparts and therefore contribute to molecular alterations observed either when intracellular protein are exposed to high doses of reducing sugars (i.e. in diabetes) or after a long time exposure (i.e. in Gzero-arrested cells during aging).

Animals↗

PubMed1996

Analysis of the solution structure of the homeodomain of rat thyroid transcription factor 1 by 1H-NMR spectroscopy and restrained molecular mechanics.

The solution structure of the rat thyroid transcription factor 1 (TTF-1) homeodomain has been elucidated by 1H-NMR and restrained modeling. The TTF-1 homeodomain folds in the same manner as classical homeodomains, with three helices, a loose loop between the first two helices, and a tight turn between helix II and helix III. The typical assembly of the hydrophobic core is maintained and N-capping motifs are identified in helix I and helix III. The N-terminal stretch of helix II exhibits some mobility, similar to the preceding loop region, which may be related to its anomalous capping. The N-terminal decapeptide and the C-terminal octapeptide of the molecule (68 residues long) are disordered. All the previous characteristics are shared by all known isolated homeodomain structures. An important difference among these structures occurs at the C-terminal extension of helix III, which is either disordered or helically folded. In the TTF-1 homeodomain, the C-terminal extension of helix III (residues 51-59) appears structured, albeit not as rigidly as the preceding portion. Analysis of the NOEs and hydrogendeuterium exchange of backbone amides provides evidence for discontinuity between the two moieties of helix III, which is introduced by a tightening or a kink of residues 51-53.

Animals↗

PubMed1996

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5'-TAAT-3'. The homeodomain of thyroid transcription factor 1 (TTF-1HD) binds to sequences containing a 5'-CAAG-3' core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF-1HD by both structural and functional approaches. As already suggested by the three-dimensional structure of TTF-1HD, the DNA binding specificities of the TTF-1, Antennapedia and Engrailed homeodomains, either wild-type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3' end of the core motif 5'-NAAN-3'. The nucleotide at the 5' position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF-1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

Animals↗

PubMed1996

In the TTF-1 homeodomain the contribution of several amino acids to DNA recognition depends on the bound sequence.

The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5'-CAAG-3' core motif. Most other homeodomains instead recognize sites containing the 5'-TAAT-3' core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5'-CAAG-3' core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5'-CAAG-3' core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5'-CAAG-3' core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5'-CAAG-3' core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases.

Amino Acids↗

PubMed1995

Expression, purification and functional characterisation of a Kunitz-type module from chicken type VI collagen.

The primary amino acid sequence of the carboxyl-terminal portion of the alpha 3 chain of chicken type VI collagen (K-VI) presents a 58-residue motif with a high degree of homology with members of the Kunitz serine-proteinase inhibitors family. This module was cloned, expressed in E. coli, purified and compared to the bovine pancreatic trypsin inhibitor (BPTI) in an inhibition profile assay of two serine proteases, trypsin and plasmin. We found that recombinant K-VI is not endowed with inhibitory activity but it slightly activates both plasmin and trypsin, differently from other members of the family. Moreover, the ability to inhibit the serine protease activity is also lacking in the intact type VI collagen molecule.

Animals↗

PubMed1995

Definition of the DNA-binding specificity of TTF-1 homeodomain by chromatographic selection of binding sequences.

The homeodomain of the thyroid transcription factor-1 (TTF-1HD) shows a peculiar DNA-binding specificity, preferentially recognizing sequences having the 5'-CAAG-3' core motif. In order to detail the DNA-binding specificity of this protein, a TTF-1HD-Sepharose column chromatography was used. A sequential selection and amplification of sequences was performed. TTF-1HD binding activity for selected and unselected sequences was measured. The presence of the 5'-CAAG-3' core motif was necessary, but not sufficient, to obtain the maximal binding activity for TTF-1HD. However, several of the selected sequences do not contain the 5'-CAAG-3' core motif and are bound by TTF-1HD only 2-fold less with respect to sequences bound with the highest affinity. Therefore, these data indicate that TTF-1HD specifically recognizes a spectrum of sequences wider than previously determined.

Base Sequence↗