Biology subjects

Chamness, L. M.

Publications and source records attributed to Chamness, L. M..

7 recordsLinked to original sources

bioRxiv2025

Deep Receptor Scanning Reveals General Sequence Constraints on GPCR Biosynthesis

G protein-coupled receptors (GPCRs) mediate a variety of signaling pathways and are the most common pharmacological targets. While advances in structural biochemistry have provided deep functional insights into key receptors, many of the 800+ human GPCRs remain understudied. We introduce a versatile "deep receptor scanning" platform that can be used to experimentally characterize 766 human GPCRs and 174 known GPCR splice variants in parallel. We use this platform to quantitatively characterize the relative abundance of canonical and alternative receptor transcripts, their translational efficiency, and the plasma membrane expression of each receptor in the context of a recombinant pool of HEK293T cells expressing individual GPCRs. We then employ machine learning to identify specific structural features that modulate GPCR expression. This experimental platform and informatic approach are compatible with a variety of assays and can be used to efficiently explore the biochemical and pharmacological properties of the GPCRome.

biochemistry↗

bioRxiv2023

Divergent Pairwise Epistasis in the Context of Unstable Membrane Protein Variants

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally. Impact StatementMany of the synergistic interactions between mutations within a gene are modified in the context of unstable proteins in a manner that depends on how the variants promote misfolding in the cell.

biochemistry↗

bioRxiv2022

Elucidation of Global Trends in the Effects of VX-661 and VX-445 on the Expression of Clinical CFTR Variants

Cystic fibrosis (CF) is a chronic genetic disease caused by mutations that compromise the expression and/ or function of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR). Most people with CF harbor a common misfolded CFTR variant ({Delta}F508), which can be rescued by combination therapies containing "corrector" compounds that restore its expression. Nevertheless, there are over 400 other CF variants that differ in their sensitivity to correctors for reasons that remain unclear. In this work, we utilize deep mutational scanning to quantitatively compare the effects of two FDA-approved correctors on the plasma membrane expression of 129 known CF variants, including 45 that are currently unclassified. Across 67 variants with attenuated expression, we find that VX-661-sensitive variants generally exhibit intermediate expression and feature mutations near its binding pocket in transmembrane domains (TMDs) 1, 2, 3, and 6. VX-445 also primarily rescues variants with intermediate expression but is instead uniquely effective towards mutations near its binding pocket in TMDs 10 & 11. Structural calculations suggest corrector binding provides similar stabilization to both sensitive and insensitive variants. These findings collectively suggest the mutation-specific effects of these compounds depend on the degree of variant destabilization and/ or the timing of cotranslational folding defects. Combining these correctors synergistically rescues variants with deficient and intermediate expression alike, presumably by doubling the total binding energy and suppressing defects at different stages of translation. These results provide an unprecedented overview of the properties of rare CFTR variants and establish new tools for CF pharmacology.

biochemistry↗

bioRxiv2022

Molecular Basis for Variations in the Sensitivity of Pathogenic Rhodopsin Variants to 9-cis-Retinal

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. 67 of these apparent class II variants exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts in relation to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants ({Delta}N73, S131P, and R135G) and show that two retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

biochemistry↗

bioRxiv2020

Disease-Linked Super-Trafficking of a Mutant Potassium Channel

Gain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. We tested whether any of the known GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface--"super-trafficking". We found that levels of R231C KCNQ1 in the plasma membrane are 5-fold higher than wild type KCNQ1. This arises from both enhanced translocon-mediated membrane integration of the S4 voltage-sensor helix and an energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 is constitutively active, but also revealed the single channel activity of this mutant to be only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the net effects of super-trafficking, reduced single channel activity, and constitutive channel activation. These investigations document membrane protein super-trafficking as a contributing mechanism to human disease.

biophysics↗

bioRxiv2019

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). Though they function at the plasma membrane, mammalian GnRHRs tend to accumulate within the secretory pathway. Their apparent instability is believed to have evolved in response to selection for attenuated GnRHR activity. Nevertheless, the structural basis of this adaptation remains unclear. We find that this adaptation coincides with a C-terminal truncation and an increase in the polarity of its transmembrane (TM) domains. This enhanced polarity compromises the translocon-mediated cotranslational folding of two TM domains. Moreover, replacing a conserved polar residue in TM6 with an ancestral hydrophobic residue partially restores GnRHR expression with minimal impact on function. An evolutionary analysis suggests variations in the polarity of this residue are associated with reproductive differences. Our findings suggest the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.

biochemistry↗

bioRxiv2019

Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamic simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip-site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results demonstrate that cotranslational folding of this protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding and/ or binding events may also stimulate PRF.

biochemistry↗