methodology-for-studying-interactions-vitamin-membrane-receptors

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...

University of Minnesota

Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor 5 networks that are highly organized.

Testis-specific serine/threonine kinase 2 (TSSK2) is an important target for reversible male contraception. A high-throughput screen of ≈17 000 compounds using a mobility shift assay identified two potent series of inhibitors having a pyrrolopyrimidine or pyrimidine core. The pyrrolopyrimidine 10 (IC 22 nm; GSK2163632A) and the pyrimidine 17 (IC 31 nm; ALK inhibitor 1) are the most potent TSSK2 inhibitors in these series, which contain the first sub-100 nanomolar inhibitors of any TSSK isoform reported, except for the broad kinase inhibitor staurosporine. The novel, potent pyrimidine TSSK2 inhibitor compound 19 (IC 66 nm; 2-[[5-chloro-2-[2-methoxy-4-(1-methylpiperidin-4-yl)anilino]pyrimidin-4-yl]amino]-N-methylbenzenesulfonamide) lacks the potential for metabolic activation. Compound 19 had a potency rank order of TSSK1>TSSK2>TSSK3>TSSK6, indicating that potent dual inhibitors of TSSK1/2 can be identified, which may be required for a complete contraceptive effect. The future availability of a TSSK2 crystal structure will facilitate structure-based discovery of selective TSSK inhibitors from these pyrrolopyrimidine and pyrimidine scaffolds.

Deepti Mudaliar

High Throughput Screening Laboratory,
Institute for Therapeutics Discovery & Development,
High Throughput Screening Laboratory, Institute for Therapeutics Discovery & Development

University of Minnesota

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Deepti Mudaliar

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}High Throughput Screening Laboratory,Institute for Therapeutics Discovery & Development,High Throughput Screening Laboratory, Institute for Therapeutics Discovery & Development

University of Minnesota

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

High Throughput Screening Laboratory,
Institute for Therapeutics Discovery & Development,
High Throughput Screening Laboratory, Institute for Therapeutics Discovery & Development