William A. Elam
C4 Therapeutics, 675 W. Kendall Street, Cambridge, MA 02142, USA
Human glucocorticoid receptor (hGR) is a cytoplasmic, chaperone-associated transcription factor. Binding to glucocorticoid (GC) promotes hGR migration to the nucleus where it functions in gene regulation. CEM 3R43 cells, leukemic lymphoblasts, contain a mutant hGR (L753F within the ligand binding domain; LBD) that exhibit temperature-sensitive binding to the GC dexamethasone (DEX), and is only able to achieve nuclear translocation and induction of apoptosis at 4°C but not 37°C. We hypothesized that naturally occurring organic osmolytes, previously known to stabilize proteins and promote the folding and function of misfolding or disordered proteins, could rescue the hGR mutant temperature-sensitivity. CEM 3R43 cell media was supplemented with trimethylamine-N-oxide (TMAO), and intracellular TMAO concentrations measured by NMR. In cell lysates, TMAO restores the ability of mutant hGR to bind DEX. However, addition of TMAO to cell media in the presence of DEX resulted in only weak induction of apoptosis, which we attribute to a TMAO-mediated stabilization of interactions between hGR and cytosolic chaperones that may inhibit hGR gene regulation.
Keywords: Chaperone, proteins, folding, regulation, sensitivity.