Elisabeth Adjadj

Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA

A change of a universally conserved leucine to valine in the DNA‐binding domain of the GATA factor AreA results in inability to activate some AreA‐dependent promoters, including that of the uapA gene encoding a specific urate–xanthine permease. Some other AreA‐ dependent promoters become able to function more efficiently than in the wild‐type context. A methionine in the same position results in a less extreme, but opposite effect. Suppressors of the AreA(Val) mutation mapping in the uapA promoter show that the nature of the base in the first position of an HGATAR (where H stands for A, T or C) sequence determines the relative affinity of the promoter for the wild‐type and mutant forms of AreA. In vitro binding studies of wild‐type and mutant AreA proteins are completely consistent with the phenotypes in vivo. Molecular models of the wild‐type and mutant AreA–DNA complexes derived from the atomic coordinates of the GATA‐1–AGATAA complex account both for the phenotypes observed in vivo and the binding differences observed in vitro. Our work extends the consensus of physiologically relevant binding sites from WGATAR to HGATAR, and provides a rationale for the almost universal evolutionary conservation of leucine at the seventh position of the Zn finger of GATA factors. This work shows inter alia that the sequence CGATAGagAGATAA, comprising two almost adjacent AreA‐binding sites, is sufficient to ensure activation of transcription of the uapA gene.

Frequency Distribution of Second Solid Cancer Locations in Relation to the Irradiated Volume Among 115 Patients Treated for Childhood Cancer

PURPOSE To provide better estimates of the frequency distribution of second malignant neoplasm (SMN) sites in relation to previous irradiated volumes, and better estimates of the doses delivered to these sites during radiotherapy (RT) of the first malignant neoplasm (FMN). METHODS AND MATERIALS The study focused on 115 patients who developed a solid SMN among a cohort of 4581 individuals. The homemade software package Dos_EG was used to estimate the radiation doses delivered to SMN sites during RT of the FMN. Three-dimensional geometry was used to evaluate the distances between the irradiated volume, for RT delivered to each FMN, and the site of the subsequent SMN. RESULTS The spatial distribution of SMN relative to the irradiated volumes in our cohort was as follows: 12% in the central area of the irradiated volume, which corresponds to the planning target volume (PTV), 66% in the beam-bordering region (i.e., the area surrounding the PTV), and 22% in regions located more than 5 cm from the irradiated volume. At the SMN site, all dose levels ranging from almost zero to >75 Gy were represented. A peak SMN frequency of approximately 31% was identified in volumes that received <2.5 Gy. CONCLUSION A greater volume of tissues receives low or intermediate doses in regions bordering the irradiated volume with modern multiple-beam RT arrangements. These results should be considered for risk-benefit evaluations of RT.

Reinvestigation of the Proteolytic Activity of Neocarzinostatin

Neocarzinostatin (NCS) is the most studied member of a family of chromoproteins secreted by a range of actinomycetes species. It has been proposed that in addition to their antitumoral activity related to the bound chromophores, this group of related proteins could be a secreted proteases superfamily. With the aim of dissecting the molecular basis of the proteolytic activity of NCS, an expression system allowing efficient expression of apo-NCS in Escherichia coli was constructed. The recombinant protein was properly folded and functional. Its histone-specific proteolytic activity was similar to the activity described for the natural protein. Further analyses unambiguously demonstrated that the proteolytic activity could be physically separated from NCS. This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant and natural NCS preparations. The histone degradation test commonly used to monitor proteolytic activity is extremely sensitive and may easily generate false-positive results. These results strongly suggest that the possible proteolytic activity of the proteins of this family should be critically reconsidered.

Phenylisoserine: A Versatile Amino Acid for the Construction of Novel β-Peptide Structures

The N-Boc O-tert-butyldimethysilyl-substituted hexa-beta-peptide methyl ester 18 was constructed from the O-TBS ether of (-)-(2R, 3S)-phenylisoserine. By NMR, it was determined that this homo beta-peptide adopts a highly stable beta-strand-type secondary structure in chloroform solution, which is stabilized by both hydrophobic interactions involving the OTBS methyl groups of residues i and i + 2, and inter-(five-membered)/intra (six-membered)-residue H-bonding interactions. These interactions are systematically repeated along the peptide chain and, thereby, operate in concert to stabilize the observed conformation of 18.

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

SummaryDynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from 13C relaxation rates R1, R2 and steady-state 13C-{1H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping Cα resonances and for 11 threonine Cβ single resonances. Except for three Cα relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C−H fragments exhibit fast (τe < 40 ps) restricted libration motions (S2=0.73 to 0.95). Global examination of the microdynamical parameters S2 and τe along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The β-ribbon comprising residues 37 to 47 is spatially restricted, with relatively large τe values in its hairpin region. The other β-ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala1 and Ala2, and the C-terminal residue Asn113, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain Cβ must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr65, Thr68, Thr81) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S2, τe and R2ex) or (Sinfslowsup2, Sinffastsup2and τslow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time τe for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1−S2)× τe is nearly independent of τe for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.

Key interactions in the immunoglobulin-like structure of apo-neocarzinostatin: Evidence from nuclear magnetic resonance relaxation data and molecular dynamics simulations

The three‐dimensional structure of apo‐neocarzinostatin (apo‐NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven‐stranded antiparallel β‐sandwich, very similar to the immunoglobulin folding domain. We investigated the backbone dynamics of apo‐NCS by 13C‐NMR relaxation measurements and molecular dynamics simulation. Model‐free parameters determined from the experimental data are compared with a 1.5‐nsec molecular simulation of apo‐NCS in aqueous solution. This comparison provides an accurate description of both local and collective movements within the protein. This analysis enabled us to correlate dynamic processes with key interactions of this β‐protein. Local motions that could be relevant for the intermolecular association with the ligand are also described.

Assignment of the protonated 13C resonances of apo-neocarzinostatin by 2D heteronuclear NMR spectroscopy at natural abundance

SummaryNearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural 13C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH2 side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, 13C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.