Restored mutant receptor:Corticoid binding in chaperone complexes by trimethylamine N-oxide.

Aaron L Miller,Betty H Johnson,Raj Kumar,E Brad Thompson,W Austin Elam,Shagufta H Khan,Didier Picard

doi:10.1371/journal.pone.0174183

Aaron L Miller, Betty H Johnson + Show 5 more

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https://doi.org/10.1371/journal.pone.0174183

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Abstract

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.

Highlights

The glucocorticoid receptor (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
The curve comparing restoration of specific Dex binding against trimethylamine N-oxide (TMAO) concentration is sigmoidal, with a slope consistent with a multistep cooperative folding effect on the ability of the GR to retain the GC under activating conditions (Fig 1A)
When incubated with TMAO, a significant improvement in cytosolic GR-specific binding of Dex is demonstrable (Fig 1B, TMAO, un-activated vs activated). These results show that in principle, TMAO can restore proper binding to the mutant GRact/l in the complex conditions of a cytosol, though the osmolyte concentrations required in vitro were high, typical of those required in the dilute solutions employed for more purified protein systems [4,6,7]

Summary

Introduction

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. 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 ̊ [2]. The hGR in these cells is a L753F mutant in its ligand binding domain (LBD) [3]. 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?

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