Molecular characterization of piebaldism in a Tunisian family.

OBJECTIVE: The present study is aimed at performing the molecular characterization of a Tunisian family with piebaldism. METHODS: As the proband and her mother showed a severe phenotype, we first chose to screen exons 10, 11, 12, 13, 16, 17 and 18 of the KIT proto-oncogene by direct sequencing. RESULTS: Direct sequencing analysis showed a C to T substitution at 1939 in exon 13 (c.1939C>T) in heterozygous state in the patient and her mother. The mutation was not found in their unaffected family members or normal controls. CONCLUSION: Our results provide additional support that mutations in the tyrosine kinase domain of the KIT gene are responsible for the severe form of piebaldism.

Insight into the structure of the pUL89 C-terminal domain of the human cytomegalovirus terminase complex.

In a previous study, we identified 12 conserved domains within pUL89, the small terminase subunit of the human cytomegalovirus. A latter study showed that the fragment pUL89(580-600) plays an important role in the formation of the terminase complex by interacting with the large terminase subunit pUL56. In this study, analysis was performed to solve the structure of pUL89(568-635) in 50% H2O/50% acetonitrile (v/v). We showed that pUL89(568-635) consists of four alpha helices, but we did not identify any tertiary structure. The fragment 580-600 formed an amphipathic alpha helix, which had a hydrophobic side highly conserved among herpesviral homologs of pUL89; this was not observed for its hydrophilic side. The modeling of pUL89(457-612) using the recognition fold method allowed us to position pUL89(580-600) within this domain. The theoretical structure highlighted three important features. First, we identified a metal-binding pocket containing residues Asp(463), Glu(534), and Glu(588), which are highly conserved among pUL89 homologs. Second, the model predicted a positively charged surface able to interact with the DNA duplex during the nicking event. Third, a hydrophobic patch on the top of the catalytic site suggested that this may constitute part of the pUL89 site recognized by pUL56 potentially involved in DNA binding.

Interlocked mismatch-aligned arrowhead DNA motifs.

BACKGROUND: Triplet repeat sequences are of considerable biological importance as the expansion of such tandem arrays can lead to the onset of a range of human diseases. Such sequences can self-pair via mismatch alignments to form higher order structures that have the potential to cause replication blocks, followed by strand slippage and sequence expansion. The all-purine d(GGA)n triplet repeat sequence is of particular interest because purines can align via G.G, A.A and G.A mismatch formation. RESULTS: We have solved the structure of the uniformly 13C,15N-labeled d(G1-G2-A3-G4-G5-A6-T7) sequence in 10 mM Na+ solution. This sequence adopts a novel twofold-symmetric duplex fold where interlocked V-shaped arrowhead motifs are aligned solely via interstrand G1.G4, G2.G5 and A3.A6 mismatch formation. The tip of the arrowhead motif is centered about the p-A3-p step, and symmetry-related local parallel-stranded duplex domains are formed by the G1-G2-A3 and G4-G5-A6 segments of partner strands. CONCLUSIONS: The purine-rich (GGA)n triplet repeat sequence is dispersed throughout the eukaryotic genome. Several features of the arrowhead duplex motif for the (GGA)2 triplet repeat provide a unique scaffold for molecular recognition. These include the large localized bend in the sugar-phosphate backbones, the segmental parallel-stranded alignment of strands and the exposure of the Watson-Crick edges of several mismatched bases.

A K cation-induced conformational switch within a loop spanning segment of a DNA quadruplex containing G-G-G-C repeats.

We have identified a unique structural transition (in slow exchange on the NMR time scale) in the tertiary fold of the d(G-G-G-C-T4-G-G-G-C) quadruplex on proceeding from Na+ to K+ as counterion in aqueous solution. Both monovalent cation-dependent conformations exhibit certain common structural features, which include head-to-tail dimerization of two symmetry-related stem-hairpin loops, adjacent strands which are antiparallel to each other and adjacent stacked G(syn).G(anti). G(syn).G(anti) tetrads in the central core of the quadruplexes. The Na and K cation stabilized structures of the d(G-G-G-C-T4-G-G-G-C) quadruplexes differ in the conformations of the T-T-T-T loops, the relative alignment of G.C base-pairs positioned opposite each other through their major groove edges and potentially in the number of monovalent cation binding sites. We have identified potential K cation binding cavities within the symmetry-related T-T-T-G segments, suggesting the potential for two additional monovalent cation binding sites in the K cation-stabilized quadruplex relative to its Na cation-stabilized counterpart. Modeling studies suggest that the major groove edges of guanine residues in Watson-Crick G.C base-pairs could potentially be bridged by coordinated K cations in the d(G-G-G-C-T4-G-G-G-C) quadruplex in KCl solution in contrast to formation of G.C.G.C tetrads for the corresponding quadruplex in NaCl solution. Our results defining the molecular basis of a Na to K cation-dependent conformational switch in the loop spanning segment of the d(G-G-G-C-T4-G-G-G-C) quadruplex may have relevance to recent observations that specific K cation coordinated loop conformations within quadruplexes exhibit inhibitory activity against HIV integrase.

NMR structure of the HIV-1 regulatory protein VPR.

The human immunodeficiency virus type 1 (HIV-1) genome encodes a highly conserved regulatory gene product, Vpr (96 residues, 14kDa), which is incorporated into virions. In the infected cells, Vpr, expressed late in the virus cycle, is believed to function in the early phases of HIV-1 replication, such as nuclear migration of pre-integration complex, transcription of the proviral genome, viral multiplication by blocking cells in G2 phase and regulation of apoptosis phenomenon. Vpr has a critical role in long term AIDS disease by inducing infection in non-dividing cells such as monocytes and macrophages. To gain insight into the structure-function relationships of Vpr, the (1-96)Vpr protein was synthesized with 22 labeled amino acids. Its 3D structure was analyzed in the presence of CD(3)CN and in pure water at low pH and refined by restrained simulated annealing. The structure of the protein is characterized by three well-defined alpha-helices: 17-33, 38-50 and 56-77 surrounded by flexible N and C-terminal domains. In contrast to the structure obtained in the presence of TFE, the three alpha-helices are folded around a hydrophobic core constituted of Leu, Ile, Val and aromatic residues as illustrated by numerous long range NOEs. This structure accounts for the interaction of Vpr with different targets.

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.

Solution structure of a Na cation stabilized DNA quadruplex containing G.G.G.G and G.C.G.C tetrads formed by G-G-G-C repeats observed in adeno-associated viral DNA.

We have applied NMR and molecular dynamics computations including intensity based refinement to define the structure of the d(G-G-G-C-T4-G-G-G-C) dodecanucleotide in 100 mM NaCl solution. The G-G-G-C sequence is of interest since it has been found as tandem repeats in the DNA sequence of human chromosome 19. The same G-G-G-C sequence is also seen as islands in adeno-associated virus, a human parvovirus, which is unique amongst eukaryotic DNA viruses in its ability to integrate site-specifically into a defined region of human chromosome 19. The d(G-G-G-C-T4-G-G-G-C) sequence forms a quadruplex in Na cation containing solution through head-to-tail dimerization of two symmetry-related stem-hairpin loops with adjacent strands antiparallel to each other around the quadruplex. The connecting T4 loops are of the lateral type, resulting in a quadruplex structure containing two internal G.G.G.G tetrads flanked by G.C.G.C tetrads. The G(anti).G(syn).G(anti).G(syn) tetrads are formed through dimerization associated hydrogen bonding alignments of a pair of Hoogsteen G(anti).G(syn) mismatch pairs, while the G(anti).C(anti).G(anti).C(anti) tetrads are formed through dimerization associated bifurcated hydrogen bonding alignments involving the major groove edges of a pair of Watson-Crick G.C base-pairs. The quadruplex contains two distinct narrow and two symmetric wide grooves with extensive stacking between adjacent tetrad planes. The structure of the quadruplex contains internal cavities that can potentially accommodate Na cations positioned between adjacent tetrad planes. Three such Na cations have been modeled into the structure of the d(G-G-G-C-T4-G-G-G-C) quadruplex. Finally, we speculate on the potential role of quadruplex formation involving G.G.G.G and G.C.G.C tetrads during the integration of the adeno-associated parvovirus into its target on human chromosome 19, both of which involve stretches of G-G-G-C sequence elements.

Sanjad-Sakati syndrome in a Tunisian child.

Sanjad-Sakati syndrome (SSS) (OMIM 241410) is a rare autosomal recessive disorder characterized by congenital hypoparathyroidism with growth and mental retardation associated with seizures and a characteristic physiognomy. SSS molecular pathology has been shown to be due to mutations in the TBCE gene on chromosome 1q42-q43. All affected patients of Arab origin are homozygous for a 12-bp (155-166del) deletion in exon 3 of this gene. We report on a Tunisian child with SSS who was homozygous for the 155-166del mutation. Our findings provide additional support of the common (155-166del) deletion founder effect in exon 3 of the TBCE gene in Arab patients. It is very likely that this mutation originated in the Middle East and was introduced in Tunisia by the Banu Hilal invaders.

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing.

Insight into protein nuclear magnetic resonance research.

Nuclear magnetic resonance (NMR) is one of the most powerful techniques to investigate the geometry of molecules in solution. It has been widely applied, in recent years, to the study of protein conformation. However, full reconstruction of the 3-D structure of such macro-molecules, still constitutes a real challenge for the spectroscopist. Skills as diverse as biology, spectroscopy, signal processing, or computer sciences, are required. This paper presents various aspects of the research in that domain, and our contribution to it.

Determination of 3J(H3í, Pi+1) and 3J(H5í/5i, Pi) coupling constants in 13C-labeled nucleic acids using constant-time HMQC.

A novel 1H-13C correlated two-dimensional experiment, CT-HMQC-J, for the measurement of three-bond proton-phosphorus coupling constants in 13C-labeled DNA is described. The experiment is based on the intensity difference of 1H-13C cross peaks in the presence and absence of the proton-phosphorus coupling interaction during the constant-time period in HMQC experiment. The 3J(H, P) coupling constants can be easily extracted from the intensity ratios of the two experiments. The method has been applied to a uniformly 13C, 15N-labeled d(GGAGGAT) 7-mer DNA sample. The proton-phosphorus coupling constants determined from CT-HMQC-J, together with the other three-bond coupling constants, are used to determine beta and epsilon torsion angles. The introduction of beta and epsilon restraints has improved the convergence as well as the quality of d(GGAGGAT) structure.

In situ immunocytochemical detection of potyviral proteins in plant cells.

A method for in situ protein immunodetection using a peroxidase labeling system is described for detecting functional and structural proteins encoded by potato virus Y (Tunisian isolate) in plant tissues. Such Potyviruses are characterized by the accumulation of inclusion bodies containing viral encoded proteins other than coat protein. These proteins are functional at early stages of infection, making them easy to detect. Data are compared to those obtained by immunofluorescence techniques. Our technique can be used as a preliminary method for rapid detection of virus infection using antibodies directed against functional proteins.

[Is there a real place for robotics in proximal tubal surgery?]. / Y a-t-il une réelle place à la robotique pour la chirurgie tubaire proximale ?

Tubal surgery requires a fine gesture. Its complexity, the difficulty of learning, the low recognition at the time of T2A and the success of Assisted Reproductive Technology (ART) could have announced the obituary of this surgery. However, in well-trained hands, tubal surgery avoids unnecessary ARTs and even allows pregnancies when medical technology fails. In this context, it is legitimate to ask whether the contribution of new technologies in the operating theatre, such as robotic surgery, can lead to an easy realization of microsurgery on a particularly complex portion of Fallopian tubes: the proximal segment.

Molecular mimicry in inducing DNA damage between HIV-1 Vpr and the anticancer agent, cisplatin.

The human immunodeficiency virus type 1 (HIV-1) viral protein R (vpr) gene is an evolutionarily conserved gene among the primate lentiviruses. Several functions are attributed to Vpr including the ability to cause cell death, cell cycle arrest, apoptosis and DNA damage. The Vpr domain responsible for DNA damage as well as the mechanism(s) through which Vpr induces this damage is unknown. Using site-directed mutagenesis, we identified the helical domain II within Vpr (aa 37-50) as the region responsible for causing DNA damage. Interestingly, Vpr Delta(37-50) failed to cause cell cycle arrest or apoptosis, to induce Ku70 or Ku80 and to suppress tumor growth, but maintained its capability to activate the HIV-1 LTR, to localize to the nucleus and to promote nonhomologous end-joining. In addition, our cytogenetic data indicated that helical domain II induced chromosomal aberrations, which mimicked those induced by cisplatin, an anticancer agent. This novel molecular mimicry function of Vpr might lead to its potential therapeutic use as a tumor suppressor.

Localization of a novel autosomal recessive non-syndromic hearing impairment locus DFNB63 to chromosome 11q13.3-q13.4.

Hereditary hearing impairment is the most genetically heterogeneous trait known in humans. So far, 50 published autosomal recessive non-syndromic hearing impairment (ARNSHI) loci have been mapped, and 23 ARNSHI genes have been identified. Here, we report the mapping of a novel ARNSHI locus, DFNB63, to chromosome 11q13.3-q13.4 in a large consanguineous Tunisian family. A maximum LOD score of 5.33 was obtained with microsatellite markers D11S916 and D11S4207. Haplotype analysis defined a 5.55 Mb critical region between microsatellite markers D11S4136 and D11S4081. DFNB63 represents the sixth ARNSHI locus mapped to chromosome 11. We positionally excluded MYO7A from being the DFNB63-causative gene. In addition, the screening of two candidate genes, SHANK2 and KCNE3, failed to reveal any disease-causing mutations.

Interaction between the HIV-1 protein Vpr and the adenine nucleotide translocator.

The HIV-1 protein Vpr circulates in the serum of seropositive individuals and in the cerebrospinal fluid of AIDS patients with neurological disorders. Vpr triggers apoptosis of numerous cell types after extracellular addition, vpr gene transfer or in the context of viral infection. Moreover, in vivo, transgenic mice over-expressing Vpr have enhanced T lymphocytes apoptosis. In previous studies, we suggested that the Vpr apoptotic activities were because of its binding to the adenine nucleotide translocator (ANT), a mitochondrial ATP/ADP antiporter. To specify this interaction, fragments of both proteins were synthesized and used in biochemical and biophysical experiments. We demonstrate here that in vitro, the (27-51) and (71-82) Vpr peptides bind to a region encompassing the first ANT intermembrane space loop and part of its second and third transmembrane helices. Computational analysis using a docking program associated to dynamic simulations enabled us to construct a three-dimensional model of the Vpr-ANT complex. In this model, the N-terminus of Vpr plunges in the ANT cavity whereas the Vpr C-terminal extremity is located at the surface of the ANT allowing possible interactions with a third partner. These results could be used to design molecules acting as pro-apoptotic Vpr analogs or as apoptosis inhibitors preventing the Vpr-ANT interaction.

NMR structure of the HIV-1 regulatory protein Vpr in H2O/trifluoroethanol. Comparison with the Vpr N-terminal (1-51) and C-terminal (52-96) domains.

The human immunodeficiency virus type 1, HIV-1, genome encodes a highly conserved regulatory gene product, Vpr (96 amino acids), which is incorporated into virions in quantities equivalent to those of the viral Gag protein. In infected cells, Vpr is believed to function during the early stages of HIV-1 replication (such as transcription of the proviral genome and migration of preintegration nuclear complex), blocks cells in G2 phase and triggers apoptosis. Vpr also plays a critical role in long-term AIDS disease by inducing viral infection in nondividing cells such as monocytes and macrophages. To gain deeper insight of the structure-function relationship of Vpr, the intact protein (residues 1-96) was synthesized. Its three-dimensional structure was analysed using circular dichroism and two-dimensional 1H- and 15N-NMR and refined by restrained molecular dynamics. In addition, 15N relaxation parameters (T1, T2) and heteronuclear 1H-15N NOEs were measured. The structure of the protein is characterized by a well-defined gamma turn(14-16)-alpha helix(17-33)-turn(34-36), followed by a alpha helix(40-48)-loop(49-54)-alpha helix(55-83) domain and ends with a very flexible C-terminal sequence. This structural determination of the whole intact Vpr molecule provide insights into the biological role played by this protein during the virus life cycle, as such amphipathic helices are believed to be involved in protein-lipid bilayers, protein-protein and/or protein-nucleic acid interactions.

1H and 15N resonance assignment and secondary structure of capsicein, an alpha-elicitin, determined by three-dimensional heteronuclear NMR.

The backbone 1H and 15N resonance assignments and solution secondary structure determination of capsicein, a protein of 98 residues with a molecular mass of 10161 Da, are presented. Capsicein belongs to the elicitin family, elicitor molecules having toxic and signaling properties that are secreted by Phytophthora fungi, responsible for the incompatible hypersensitive reaction of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The protein was uniformly labeled with 15N to overcome spectral overlap of the proton resonances. A combination of 3D HOHAHA-HMQC and 3D NOESY-HMOC experiments allowed the identification of spin systems with through-bond correlations, which were in turn correlated by through-space connections. The sequential assignment was obtained for main- and side-chain resonances and led to the identification of all secondary structures. A 3D HMQC-NOESY-HMQC experiment was performed which characterized the NH(i)-NH(i+1) connections specific to alpha-helical structures. This proved particularly useful for the assignment of degenerate amide protons of successive residues in alpha-helical structures. The data show five alpha-helical regions comprising residues 5-18, 26-33, 44-58, 59-67, and 86-98 and a two-stranded antiparallel beta-sheet involving residues 70-75 and 80-85, packed around a hydrophobic core grouping all of the aromatic residues. The C-terminal secondary structure motifs of capsicein evoke phospholipase structural features, which suggests that elicitins might interact with the lipidic molecules of the plasma membrane.

Target specificity of human immunodeficiency virus type 1 NCp7 requires an intact conformation of its CCHC N-terminal zinc finger.

The modification of zinc-binding residues inside the conserved CCHC motif of human immunodeficiency virus type 1 NCp7, in particular into CCHH, induces a complete loss of infectivity. Since the mutant His28NCp7 has been shown to be devoid of infectivity in vivo, the structure-function relationships of the mutant His28(12-53)NCp7 were investigated by nuclear magnetic resonance and surface plasmonic resonance. Although the Cys28-->His mutation modifies drastically the structure of the core domain (residues 12 to 53) of NCp7, His28(12-53)NCp7 still interacts with a 10-fold-lower affinity to specific nucleic acid targets, such as SL3, a stem-loop critically involved in viral RNA packaging, and without affinity change with the nonspecific, single-stranded nucleic acid poly(T). Moreover, His28(12-53)NCp7 and native (12-53)NCp7 displayed the same affinity with reverse transcriptase, but the natures of the complexes are probably different, accounting for the drastic reduction in the amount of RNA packaged in the mutated virus. We propose a structural model of His28(12-53)NCp7 that provides insights into the NCp7 structural features necessary for target recognition and that shows that the specific native structure of the zinc finger domain is strictly required for the optimal target selectivity of NCp7.

Bombyx mori single repeat telomeric DNA sequence forms a G-quadruplex capped by base triads.

A combined NMR-molecular dynamics approach has been applied to determine the solution structure of a truncated analogue of the Bombyx mori telomeric d(TTAGG) single repeat sequence in Na+ cation-containing aqueous solution. The two-fold symmetric four-stranded d(TAGG) quadruplex contains two adjacent G(syn).G(syn).G(anti).G(anti) G-tetrads sandwiched between novel (T.A).A triads with individual strands having both a parallel and antiparallel neighbour around the quadruplex. The (T.A).A triad represents the first experimental verification of a base triad alignment which constitutes a key postulate in the recently proposed model of triad-DNA. Further, the (T.A).A triad is generated by positioning an A residue through hydrogen bonding in the minor groove of a Watson-Crick T.A base pair and includes a T-A platform related to an A-A platform recently observed in the structure of the P4-P6 domain of the Tetrahymena self splicing group I ribozyme. The novel architecture of the truncated Bombyx mori quadruplex structure sets the stage for the design and potential identification of additional base tetrads and triads that could participate in pairing alignments of multi-stranded DNA structures during chromosome association and genetic recombination.