ABSTRACT
Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR) separate from their mutual binding. Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain α-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. By focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.
Subject(s)
Intrinsically Disordered Proteins/chemistry , Osmotic Pressure , Protein Folding , Animals , Circular Dichroism , Intrinsically Disordered Proteins/metabolism , Mice , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, SecondaryABSTRACT
BACKGROUND/AIMS: To test the activity of novel hydroxyvitamin D(3) analogs (20(OH)D(3), 20,23(OH)(2)D and 1,20(OH)(2)D(3)) on normal and malignant melanocytes in comparison to 1,25(OH)(2)D(3). MATERIALS AND METHODS: Human epidermal melanocytes and human and hamster melanoma cells were used to measure effects on proliferation and colony formation in monolayer and soft agar. Cell morphology and melanogenesis were also analyzed. QPCR was used to measure gene expression. RESULTS: Novel secosteroids inhibited proliferation and colony formation by melanoma cells in a similar fashion to 1,25(OH)(2)D(3), having no effect on melanogenesis. These effects were accompanied by ligand-induced translocation of VDR to the nucleus. In normal melanocytes 1α-hydroxyderivatives (1,25(OH)(2)D(3) and 1,20(OH)(2)D(3)) had stronger anti-proliferative effects than 20(OH)D(3) and 20,23(OH)(2)D(3), and inhibited dendrite formation. The cells tested expressed genes encoding VDR and enzymes that activate or inactivate vitamin D(3). CONCLUSION: Novel secosteroids show potent anti-melanoma activity in vitro with 20(OH)D(3) and 20,23(OH)(2)D(3) being excellent candidates for pre-clinical testing.
