Biomedical subjects

Joost Van Durme

Publications and source records attributed to Joost Van Durme.

5 recordsLinked to original sources

PubMed2006

GRIS: glycoprotein-hormone receptor information system.

The glycoprotein-hormone receptor information system (GRIS) presents a comprehensive view on all available molecular data for the lutropin/choriogonadotropin receptor, follitropin receptor, and thyrotropin receptor G protein-coupled receptors. It features a mutation database presently containing 696 point mutations, combined with all sequences and the associated homology models. The mutation information was automatically extracted from the literature and manually augmented with respect to constitutivity, surface expression, sensitivity to hormones, and binding affinity. All information in this integrated system is presented in a G protein-coupled receptor specialist-friendly way. A series of interactive tools such as rotamer analysis, mutation prediction, or cavity visualization aids with the design and interpretation of experiments. A universal residue numbering system has been introduced to ease database searches as well as the use of the information in conjunction with literature data from diverse origins. Users can upload new mutations. GRIS is freely accessible at http://gris.ulb.ac.be/.

Amino Acid Sequence↗

PubMed2005

Repulsive separation of the cytoplasmic ends of transmembrane helices 3 and 6 is linked to receptor activation in a novel thyrotropin receptor mutant (M626I).

Ligand-dependent activation of G protein-coupled receptors (GPCRs) involves repositioning of the juxtacytoplasmic ends of transmembrane helices TM3 and TM6. This concept, inferred from site-directed spin labeling studies, is supported by chemical cross-linking of the cytoplasmic ends of TM3 and TM6 blocking GPCR activation. Here we report a novel constitutive active mutation (M626I) in TM6 of the TSH receptor (TSHR), identified in affected members of a family with nonautoimmune hyperthyroidism. The specific constitutive activity of M626I, measured by its basal cAMP generation corrected for cell surface expression, was 13-fold higher than that of wild-type TSHR. Homology modeling of the TSHR serpentine domain based on the rhodopsin crystal structure suggests that M626 faces the side chain of I515 of TM3 near the membrane-cytoplasmic junction. Steric hindrance of the introduced isoleucine by I515 is consistent with the fact that shorter or more flexible side chains at position 626 did not increase constitutivity. Furthermore, a reciprocal mutation at position 515 (I515M), when introduced into the M626I background, acts as revertant mutation by allowing accommodation of the isoleucine sidechain at position 626 and fully restoring the constitutive activity to the level of wild-type TSHR. Thus, repulsive separation of the juxtacytoplasmic TM6 and TM3 in the M626I model conclusively demonstrates a direct link between the opening of this cytoplasmic face of the receptor structure and G protein coupling.

Amino Acid Substitution↗

PubMed2004

Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity.

An experimental murine model of Graves' disease was used to produce monoclonal antibodies (mAbs) with thyroid stimulating activity. Two of these, IRI-SAb2 and IRI-SAb3, showed particularly high potency (in the low nanomolar range) and efficacy. IRI-SAb2 behaved as a full agonist of the human TSH receptor (TSHr), even when tested in physiological salt concentrations. Both IRI-SAb2 and IRI-SAb3 were displaced from the TSHr by autoantibodies from patients with Graves' disease or harboring thyroid-blocking antibodies, but not from control subjects or patients with Hashimoto thyroiditis. The epitopes of IRI-SAb2 and IRI-SAb3 were precisely mapped, at the amino acid level, to the amino-terminal portion of the concave portion of the horseshoe structure of TSHr ectodomain. They overlap closely with each other and, surprisingly, with the epitope of a mAb with blocking activity. When injected iv in mice, both mAbs caused biological and histological signs of hyperthyroidism. Unexpectedly, they also triggered an inflammatory response in the thyroid glands. Delineation of the conformational epitopes of these stimulating antibodies opens the way to the identification of the molecular mechanisms implicated in the activation of the TSHr.

Amino Acid Sequence↗

PubMed2004

A family-based approach reveals the function of residues in the nuclear receptor ligand-binding domain.

Literature studies, 3D structure data, and a series of sequence analysis techniques were combined to reveal important residues in the structure and function of the ligand-binding domain of nuclear hormone receptors. A structure-based multiple sequence alignment allowed for the seamless combination of data from many different studies on different receptors into one single functional model. It was recently shown that a combined analysis of sequence entropy and variability can divide residues in five classes; (1) the main function or active site, (2) support for the main function, (3) signal transduction, (4) modulator or ligand binding and (5) the rest. Mutation data extracted from the literature and intermolecular contacts observed in nuclear receptor structures were analyzed in view of this classification and showed that the main function or active site residues of the nuclear receptor ligand-binding domain are involved in cofactor recruitment. Furthermore, the sequence entropy-variability analysis identified the presence of signal transduction residues that are located between the ligand, cofactor and dimer sites, suggesting communication between these regulatory binding sites. Experimental and computational results agreed well for most residues for which mutation data and intermolecular contact data were available. This allows us to predict the role of the residues for which no functional data is available yet. This study illustrates the power of family-based approaches towards the analysis of protein function, and it points out the problems and possibilities presented by the massive amounts of data that are becoming available in the "omics era". The results shed light on the nuclear receptor family that is involved in processes ranging from cancer to infertility, and that is one of the more important targets in the pharmaceutical industry.

Amino Acids↗

PubMedDate not supplied

Statistical analysis of tablet breakability methods.

PURPOSE: Using a model tablet, the influence of breakability methodology on mass uniformity of half- and quarter-tablets as well as the comparison of different data acquisition and evaluation approaches were investigated. Moreover, different breakability evaluation criteria were compared based upon distribution as well as distribution-free models. METHODS: A cross-scored tablet, i.e. having two break-marks, was broken by different methods by different persons, and the masses determined for the whole (unbroken) tablets, the half-tablets and quarter-tablets. RESULTS: Beside the possible interaction between the methodology and the person breaking the tablets, the major factor significantly influencing the mass uniformity of broken tablets is the breakability methodology. The best results, i.e. smallest loss and smallest variability, are obtained when the breaking force applied by the thumbs is directed towards the score side of the tablet, i.e. by "opening" the score. Using our model tablet, significant differences between the different evaluation criteria are observed, with the USP/NF approach being best in line with the detailed analysis of all broken tablets. CONCLUSIONS: Assuming that for this model tablet the variance is a linear function of the break-line length, the standard deviation of quarter-tablets is theoretically calculated to be 0.87 times the standard deviation of the half-tablets. As the absolute standard deviation, expressed in mass units, will thus remain approximately identical, the relative standard deviation will nearly double as the mean mass of the quarter-tablets will be half of the mean mass of the half-tablets. This was experimentally confirmed.

Analysis of Variance↗