Relax, Cool Down and Scaffold: How to Restore Surface Expression of Folding-Deficient Mutant GPCRs and SLC6 Transporters

Abstract

Many diseases arise from mutations, which impair protein folding. The study of folding-deficient variants of G protein-coupled receptors and solute carrier 6 (SLC6) transporters has shed light on the folding trajectory, how it is monitored and how misfolding can be remedied. Reducing the temperature lowers the energy barrier between folding intermediates and thereby eliminates stalling along the folding trajectory. For obvious reasons, cooling down is not a therapeutic option. One approach to rescue misfolded variants is to use membrane-permeable orthosteric ligands. Antagonists of GPCRs are—in many instances—effective pharmacochaperones: they restore cell surface expression provided that they enter cells and bind to folding intermediates. Pharmacochaperoning of SLC6 transporters is less readily achieved because the ionic conditions in the endoplasmic reticulum (ER) are not conducive to binding of typical inhibitors. The second approach is to target the heat-shock protein (HSP) relay, which monitors the folding trajectory on the cytosolic side. Importantly, orthosteric ligands and HSP-inhibitors are not mutually exclusive. In fact, pharmacochaperones and HSP-inhibitors can act in an additive or synergistic manner. This was exemplified by rescuing disease-causing, folding-deficient variants of the human dopamine transporters with the HSP70 inhibitor pifithrin-μ and the pharmacochaperone noribogaine in Drosophila melanogaster.

Rescue of two trafficking-defective variants of the neuronal glycine transporter GlyT2 associated to hyperekplexia

“…These compounds inhibit monoamine transporters by blocking the proteins in inward-open conformation and facilitate the maturation acting as pharmacochaperones. This action has led to the proposal that, during folding, SLC6 transporters have to adopt the inwardly-directed conformation for the acquisition of the native folding (Freissmuth et al 2018;Bhat et al, 2019;Asjad et al, 2017). For GlyT2, instead, these compounds decreased surface levels although the GlyT2 fraction in the membrane of bupropion or ibogaine-treated cells, despite being inferior, had the same ability to transport glycine as the membrane fraction of untreated cells.…”

Rescue of two trafficking-defective variants of the neuronal glycine transporter GlyT2 associated to hyperekplexia

“…These compounds inhibit monoamine transporters by blocking the proteins in inward-open conformation and facilitate the maturation acting as pharmacochaperones. This action has led to the proposal that, during folding, SLC6 transporters have to adopt the inwardly-directed conformation for the acquisition of the native folding (Freissmuth, Stockner, and Sucic 2018; Bhat, Newman, and Freissmuth 2019; Asjad et al 2017). For GlyT2, instead, these compounds decreased surface levels although the GlyT2 fraction in the membrane of bupropion or ibogaine-treated cells, despite being inferior, had the same ability to transport glycine as the membrane fraction of untreated cells.…”

“…As NAG is likely to reach the ER lumen due to its structure (Burstein 2018), the inhibition of CNX action could be due to the direct binding of the compound to GlyT2, an interaction that may somehow block or compete with CNX assistance. Transport inhibitors trapping the transporters in a certain conformation, may exert its action by this mechanism of pharmacochaperoning (Bhat, Newman, and Freissmuth 2019; Asjad et al 2017). Our data do not exclude NAG acts directly on the transporter conformation, although the fact that the optimal NAG concentration for increasing GlyT2 expression and function (10 μM) was much lower than the IC 50 for GlyT2 inhibition (67 ± 23 μM), conflicts this possibility.…”

Allosteric Regulation of the Follicle-Stimulating Hormone Receptor

“…During the last two decades, a number of activating and inactivating mutations, single nucleotide polymorphisms, and spliced variants of the FSHR gene have been reported in selected infertile cases (54)(55)(56). Further, structural alterations provoked by mutation can lead to abnormal misfolding and may lead to retention/ degradation of membrane proteins including GPCRs resulting in diseases (57). Several strategies including pharmacological intervention through pharmacochaperones offer a promising method to correct the defective misdirected protein to the plasma membrane.…”