Study of the RET gene and his implication in thyroid cancer: Morocco case family.

BACKGROUND: Multiple endocrine neoplasia type 2A (MEN 2A) is an autosomal dominant inherited cancer syndrome that affects multiple tissues derived from the neural crest. Inheritance of MTC is related to the presence of specific mutations in the RET proto-oncogene. Almost all mutations in MEN 2A involve one of the cysteines in the extracellular domain of the RET receptor. AIMS: The objective of the present study was the biochemical and molecular characterization of the first Moroccan clinically established MEN 2A patient and at-risk family members. SETTINGS AND DESIGN: This is a study on a family presented with MTC referred to our institute in 2004. MATERIALS AND METHODS: Peripheral blood leukocyte DNA samples were isolated and amplified by polymerase chain reaction followed by restriction enzyme analysis and DNA sequencing. RESULTS: We identified a heterozygous germ line missense mutation at codon 634 of exon 11 in the RET gene that causes a cysteine to arginine amino acid substitution in the DNA of the proband; this mutation was not found in the DNA of the parents or relatives. CONCLUSIONS: The detection of mutated MEN 2A gene carriers enables us to differentiate high-risk members from those who bear the wild-type gene. Occasionally, application of RET proto-oncogene testing may lead to the detection of unexpected de novo mutation that could be transmitted to children.

Self-association and domains of interactions of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by analytical ultracentrifugation and NMR experiments in trifluoroethanol/H(2)O mixtures.

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.

An NMR study of d(CTACTGCTTTAG).d(CTAAAGCAGTAG) showing hydration water molecules in the minor groove of a TpA step.

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-H1'(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment (approximately 3.9A) and a sudden widening at the T10:A15 base-pair (approximately 5.3A), the standard B-DNA distance being approximately 5A. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n> or =2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for A11 ending the 5'TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the A11:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to A11 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.

Synthesis and FTIR conformational studies of peptides from the basic region of c-Jun: a critical analysis on the basis of CD and NMR data.

The peptide (35 residues) corresponding to the basic subdomain (bSD) of c-Jun (residues 252-281) and its fragments NP (N-terminal peptide, 1-19) and CP (C-terminal peptide, 16-35) were synthesized in stepwise solid-phase using the tert-butyloxycarbonyl/benzyl strategy. In a previous paper, we have shown that during its binding to the DNA site CRE (cAMP-responsive element) the bSD structure was converted into alpha-helix from an initial random coil conformation [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 231, 370-380 (1995)]. Our results suggested both a high flexibility and a helical potential in bSD, these two properties seeming crucial for the accommodation of the basic subdomain of c-Jun to its specific DNA targets. In this work, we assessed the conformational variability of bSD through the study of the secondary structures of its NP and CP fragments in trifluoroethanol (TFE)/2H2O mixtures, using Fourier transform infrared (FTIR) spectroscopy. The IR results were critically analyzed in light of our previously reported circular dichroism (CD) and NMR data [Krebs, D., Dahmani, B., Monnot, M., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem, 235, 699-712 (1996)]. Upon addition of TFE, the relative areas of the seven components of the amide I. band (1700-1620 cm-1) reflected the conversion of a large amount of random coil conformation into alpha-helix for the two fragments and bSD. This effect was accompanied by more subtle variations of the less populated structures, in agreement with the results of CD and NMR experiments. The IR results stipulated the conservation of the parent bSD secondary structures in both fragments; however, NP and CP peptides did not display similar random-to-alpha-helix stabilization pattern upon additions of TFE to aqueous solutions. The profile from CD signal at 222 nm was found sigmoidal for NP and almost linear for CP, while that corresponding to the parent peptide bSD was just in between those of its fragments. Thus, the present study confirms the high flexibility and helix propensity of the c-Jun basic subdomain and suggests that the N- and C-terminal parts of the peptide do not follow the same random-to-helix conversion profile during their complexation with DNA.

The basic subdomain of the c-Jun oncoprotein. A joint CD, Fourier-transform infrared and NMR study.

The structural properties of the basic subdomain of the basic zipper (bZIP) protein c-Jun were examined by joint means of 1H-NMR, CD and Fourier-transform infrared (FTIR) spectroscopies. The basic subdomain (residues 252-281 in c-Jun) is responsible for sequence-specific recognition of DNA. A modified basic subdomain bSD (residues 1-35) and its N-terminal part and C-terminal part fragments (NP, residues 1-19; and, CP, residues 16-35) were prepared by solid-phase synthesis and purified by HPLC. In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% alpha helix. However, upon mixing bSD and a specific DNA fragment, i.e. a CRE(cAMP-responsive element)-containing hexadecanucleotide, the alpha helix was stabilized to an extent of 20% at 20 degrees C or 35% at 2 degrees C. At the same time, no significant change could be detected in the DNA spectra. Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the alpha-helix content so that about 60% of alpha helix was found at a ratio of 75% trifluoroethanol (20 degrees C). These effects were reflected in both CD and FTIR measurements. Changes shown by the CD spectra during the process suggested a mechanism dominated by a two-state helix/unordered transition. NMR data, namely alpha H chemical shifts, NOE cross-peaks and NH temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c-Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution. Trifluoroethanol stabilized the alpha-helix structure mainly at these four sites. The malleability of the basic subdomain of c-Jun was emphasized in relation to its ability to fit the DNA helix in adopting an alpha-helix structure. The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide.

Sequence dependent effects of CpG cytosine methylation. A joint 1H-NMR and 31P-NMR study.

The impact of cytosine methylation in the central CpG step of two closely related octanucleotide duplexes d(CATCGATG)2 and d(CTTCGAAG)2 was examined by 1H-NMR and 31P-NMR experiments, and a quantitative structural analysis was performed using the NOE-derived distances, the sugar puckers and the epsilon torsion angles. The two starting oligonucleotides displayed a B-DNA conformation with, however, significant local structure differences. Although the methylated oligonucleotides retained their B-DNA conformation, different structural and thermal stability effects were observed. The magnitude of the methylation effects was to depend on the initial conformation of the CpG site, which is governed by the nature of the dinucleotide AT or TT located on the CpG flanks. As an example of sequence dependence, the methylation of CpG entailed larger conformational variation in d(CATCGATG)2 than in d(CTTCGAAG)2. In this study, the 1H and 31P chemical-shift parameters averred as extremely sensitive probes for detecting subtle conformational changes. Finally, our comparative results may aid our understanding of the structural and related biological effects produced by cytosine methylation in DNA.

Effect of distortions in the phosphate backbone conformation of six related octanucleotide duplexes on CD and 31P NMR spectra.

We examined the structural properties of six octanucleotide duplexes d(TGACGTCA), d(ACTGCAGT), d(CTTCGAAG), d(CATCGATG), d(GTACGTAC), and d(CATGCATG). Circular dichroism (CD) and 2D 31P and 1H NMR spectroscopies were used in conjunction. Although of the B-DNA type, it was possible to arrange CD spectra into two families, A and B. Family A resembled poly(dG-dC) with a positive signal at approximately 280 nm and a negative one at approximately 260 nm, while family B resembled poly(dA-dT) with a positive signal at approximately 270 nm and a negative one at approximately 250 nm. All 31P resonances were assigned through constant-time heteronuclear 31P-1H correlated spectra. J(H3'-P) coupling constants related to dihedral angeles epsilon (C4'-C3'-O3'-P) were determined from 1H-31P J-resolved selective proton-flip 2D experiments. A good correlation was observed between 31P chemical shifts and coupling constants for all oligonucleotides. The patterns of these two parameters vs the base position along the sequences were almost similar. They were confronted with CD spectra. The results indicated that the position and magnitude of the signals were mainly affected by the CpG and ApT steps whose 31P chemical shifts were the farthest away from the mean 31P chemical shift value. This is in keeping with greater rigidity at these steps and should explain the influence of the local order on the shape of the CD spectra. Lastly, both UV absorption and 31P chemical shifts vs temperature provided normal temperature melting (Tm) values for all of the octanucleotide duplexes except for d(CTTCGAAG), for which the Tm was approximately 10 degrees C lower compared to its counterpart d(CATCGATG). The decrease in the thermal stability of this octanucleotide duplex was imputed to its contained TT and AA repeats, which might be able to induce correlated base destacking and phosphate group distortion in the oligonucleotide and especially on the intermediate CpG. We demonstrate that the CpG step displayed 31P NMR properties similar to those found in mismatched nucleotides exclusively in the d(CTTCGAAG) duplex.

Structural deviations at CpG provide a plausible explanation for the high frequency of mutation at this site. Phosphorus nuclear magnetic resonance and circular dichroism studies.

CpG sites in DNA are hotspots for mutations leading to human genetic disorders. However, the structural basis for these events were still unclear and necessitated a deeper evaluation. Our experiments with phosphorus-31 nuclear magnetic resonance, ultraviolet-melting and circular dichroism on two related CpG-containing octanucleotide duplexes show that CpG is a malleable step whose conformation and thermal stability are strongly dependent on the nature of its flanking steps. We conclude that the CpG step may exert a deleterious structural influence on the helix very much like the mismatch containing steps. This peculiar property of CpG should constitute a molecular basis for its recognition by various ligands as well as for mutations affecting CpG and hence an explanation for its rarity in vertebrate genomes.

Alteration of heme axial ligands in hemoglobin by organic solvents analyzed by CD, FTIR, and XANES techniques.

The effects of mixed solvents on the ligand binding site in hemoglobin have been investigated though three spectroscopic techniques. Two classes of organic solvents (amides and alcohols) known to increase or decrease the hemoglobin affinity have been chosen for this study. The analysis of the iron CO stretching band shows that the ligand binding sites of alpha CO and beta CO subunits inside the alpha 2 beta 2 hemoglobin tetramer exhibit multiple conformations. From the circular dichroism and X-ray absorption near-edge structure data, it appears that no core deformation or heme reorientation occur with the affinity changes. The iron-ligand average bond angle is the sole parameter that depends on the external solvent. Since cosolvents seem to affect the dynamics rather than the hindrance of the heme cavity, we suggest that the protein affinity could be associated with a hierarchy of subtle dynamic states.

Relationship between protein/solvent proton exchange and progressive conformation and fluctuation changes in hemoglobin.

The pH dependence of the tertiary structural changes and the quaternary reorganization of the alpha beta interface of oxyhemoglobin in solution has been previously observed in our laboratory. The present work aims to establish whether or not these progressive structural changes with pH result from proton exchange between the protein and the solvent. We therefore have used infrared spectroscopy, acid/base titration and 1H/2H exchange to assess the effect of external proton concentration on the structural and dynamic properties of the hemoglobin molecule in solution. This study is also performed on the carbonmonoxy form since the affinity of hemoglobin for CO is much higher than for oxygen. The present findings demonstrate that affinity changes are closely related to protein fluctuations, as well as structural modifications. An increased affinity, induced either by ligand replacement or pH variation, is associated with the constrained chains exhibiting a lower degree of fluctuation, together with a looser alpha beta interface association.

Implication of the alpha 1 beta 1 interface in the hemoglobin affinity changes. A comparative study between normal and San Diego fully ligated hemoglobins.

The alpha 1 beta 1 interface of normal and mutated San Diego hemoglobins in their fully liganded form was investigated, through the SH vibrational absorption of beta-112 cysteine, by Fourier-transform infrared spectroscopy. The center frequency of this thiol group was significantly shifted in San Diego hemoglobin compared with normal human hemoglobin. Different dimer organization between the two proteins was also revealed by circular dichroism of the heme. These findings agree well with assessment that the alpha 1 beta 1 interface, far from being inert, is involved in the affinity changes of the hemoglobin molecule.