Date Published: March 16, 2017
Publisher: Public Library of Science
Author(s): Aaron L. Miller, W. Austin Elam, Betty H. Johnson, Shagufta H. Khan, Raj Kumar, E. Brad Thompson, Didier Picard.
Without a glucocorticoid (GC) ligand, the transcription factor glucocorticoid receptor (GR) is largely cytoplasmic, with its GC-binding domain held in high affinity conformation by a cluster of chaperones. Binding a GC causes serial dis- and re-associations with chaperones, translocation of the GR to the nucleus, where it binds to DNA sites and associates with coregulatory proteins and basic transcription complexes. Herein, we describe the effects of a potent protective osmolyte, trimethylamine N-oxide (TMAO), on a conditions-dependent “activation-labile” mutant GR (GRact/l), which under GR-activating conditions cannot bind GCs in cells or in cell cytosols. In both cells and cytosols, TMAO restores binding to GRact/l by stabilizing it in complex with chaperones. Cells bathed in much lower concentrations of TMAO than those required in vitro show restoration of GC binding, presumably due to intracellular molecular crowding effects.
The GR is a GC-driven transcription factor located mostly in the cytoplasm, where it is bound with several chaperone proteins which keep it in a configuration favorable for binding GC ligands. Binding a GC causes a series of dynamic interchanges of chaperones, culminating in a move to the nucleus, where GR binds to DNA to regulate transcription at specific genes . Since in leukemic lymphoblasts the GC-GR mechanism drives cell apoptosis, we used this as a selective mechanism, isolating clone CEM 3R43 of GR-resistant cells, which bind and retain the GC dexamethasone (Dex) at 4°C but not at 37° . The hGR in these cells is a L753F mutant in its ligand binding domain (LBD) . Thus both the cells and the mutant protein have been termed activation-labile (act/l). Could function be restored to this temperature-and salt-sensitive mutation by use of stabilizing osmolytes?
TMAO and other “protective” organic osmolytes exist in a wide variety of eukaryotic systems, where they act to prevent denaturation of cellular proteins from extremes of temperature, dehydration, osmotic stresses and other potentially harmful conditions. The genes responsible for production of these compounds are under strict control . Protective osmolytes act through the balance of aquaphobic and aquaphilic effects on protein backbone and amino acid side chains and can restore misfolded or unfolded proteins to their normal, functional conformations . When applied to steroid hormone receptors, protective osmolytes cause folding of their ID N-terminal domains [6,12,13]. SHRs must bind certain other proteins to function, and such binding is restored or greatly enhanced in SHR N-terminal domains treated with protective osmolytes.