Towards generalizable predictions for G protein-coupled receptor variant expression.

Missense mutations that compromise the plasma membrane expression (PME) of integral membrane proteins are the root cause of numerous genetic diseases. Differentiation of this class of mutations from those that specifically modify the activity of the folded protein has proven useful for the development and targeting of precision therapeutics. Nevertheless, it remains challenging to predict the effects of mutations on the stability and/ or expression of membrane proteins. In this work, we utilize deep mutational scanning data to train a series of artificial neural networks to predict the PME of transmembrane domain variants of G protein-coupled receptors from structural and/ or evolutionary features. We show that our best-performing network, which we term the PME predictor, can recapitulate mutagenic trends within rhodopsin and can differentiate pathogenic transmembrane domain variants that cause it to misfold from those that compromise its signaling. This network also generates statistically significant predictions for the relative PME of transmembrane domain variants for another class A G protein-coupled receptor (ß2 adrenergic receptor) but not for an unrelated voltage-gated potassium channel (KCNQ1). Notably, our analyses of these networks suggest structural features alone are generally sufficient to recapitulate the observed mutagenic trends. Moreover, our findings imply that networks trained in this manner may be generalizable to proteins that share a common fold. Implications of our findings for the design of mechanistically specific genetic predictors are discussed.

Classification of the Molecular Defects Associated with Pathogenic Variants of the SLC6A8 Creatine Transporter.

More than 80 loss-of-function (LOF) mutations in the SLC6A8 creatine transporter (hCRT1) are responsible for cerebral creatine deficiency syndrome (CCDS), which gives rise to a spectrum of neurological defects, including intellectual disability, epilepsy, and autism spectrum disorder. To gain insight into the nature of the molecular defects caused by these mutations, we quantitatively profiled the cellular processing, trafficking, expression, and function of eight pathogenic CCDS variants in relation to the wild type (WT) and one neutral isoform. All eight CCDS variants exhibit measurable proteostatic deficiencies that likely contribute to the observed LOF. However, the magnitudes of their specific effects on the expression and trafficking of hCRT1 vary considerably, and we find that the LOF associated with two of these variants primarily arises from the disruption of the substrate-binding pocket. In conjunction with an analysis of structural models of the transporter, we use these data to suggest mechanistic classifications for these variants. To evaluate potential avenues for therapeutic intervention, we assessed the sensitivity of these variants to temperature and measured their response to the proteostasis regulator 4-phenylbutyrate (4-PBA). Only one of the tested variants (G132V) is sensitive to temperature, though its response to 4-PBA is negligible. Nevertheless, 4-PBA significantly enhances the activity of WT hCRT1 in HEK293T cells, which suggests it may be worth evaluating as a therapeutic for female intellectual disability patients carrying a single CCDS mutation. Together, these findings reveal that pathogenic SLC6A8 mutations cause a spectrum of molecular defects that should be taken into consideration in future efforts to develop CCDS therapeutics.

Ribosomal Frameshifting Selectively Modulates the Assembly, Function, and Pharmacological Rescue of a Misfolded CFTR Variant.

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex (EMC), which was previously found to modulate ribosome collisions during "preemptive quality control" of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

Molecular basis for the evolved instability of a human G-protein coupled receptor.

Membrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Although they function at the plasma membrane, mammalian GnRHRs accumulate within the secretory pathway. Their apparent instability is believed to have evolved through selection for attenuated GnRHR activity. Nevertheless, the molecular basis of this adaptation remains unclear. We show that adaptation coincides with a C-terminal truncation that compromises the translocon-mediated membrane integration of its seventh transmembrane domain (TM7). We also identify a series of polar residues in mammalian GnRHRs that compromise the membrane integration of TM2 and TM6. Reverting a lipid-exposed polar residue in TM6 to an ancestral hydrophobic residue restores expression with no impact on function. Evolutionary trends suggest variations in the polarity of this residue track with reproductive phenotypes. Our findings suggest that the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.