Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.
Ceramide is the simplest molecule in the class of glycosphingolipids composed of a sphingosine backbone and acyl moiety. It plays significant roles in cell signaling, apoptosis, binding of hormones, toxins, and viruses, and many other biologically important functions. Sphingomyelin, ceramide with a phosphotidylcholine head group, is another biologically vital lipid present in the myelin sheath of nerve cell axon. Regions with high concentrations of ceramide can be formed in biological membranes composed of sphingomyelin by enzymatic catalysis with sphingomyelinase. In order to better understand the biophysical and thermodynamic properties of these molecules and their mixtures, we have preformed NPT molecular dynamics simulations of hydrated 16:0 sphingomyelin bilayers with increasing concentrations of 16:0 ceramide at 323 K, 332 K, 340 K, and 358 K. From analyses of electron densities, hydrogen bonding, NMR order parameters, partial molecular volume, and partial molecular area, we have identified possible structural changes corresponding to liquid ordered and liquid disordered phases. These structural changes are the results of changes in intra and inter molecular hydrogen bonds between SM and Cer molecules. Our results correspond to DSC experiments for sphingomyelin bilayer concentrations up to 50% Cer. Above 50% concentration, we observe conformational changes in SM headgroup similar to that of the umbrella model for lipid cholesterol mixtures.
Short peptides featuring a tetrahydropyridazinedione (tpd) backbone tether exhibit reduced conformational flexibility external to the heterocyclic constraint. Analysis by NMR, molecular modeling and X-ray crystallography suggests both covalent and non-covalent stabilization of extended peptide conformations. An efficient solid-phase protocol was developed for the synthesis of a new class of β-strand mimics based on oligomeric tpd subunits.
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