Biology subjects

Penn, W. D.

Publications and source records attributed to Penn, W. D..

10 recordsLinked to original sources

bioRxiv2025

Feedback from the Nascent Chain Triggers Ribosomal Frameshifting and Transcript Decay

Though ribosomes have several features that help them maintain their reading frame, these safeguards can be bypassed by RNA structures that promote -1 programmed ribosomal frameshifting (-1PRF). We recently found that conformational transitions in the nascent polypeptide can enhance -1PRF, though its unclear whether this feedback plays a general role in translational recoding. Here we demonstrate that the translocation of nascent transmembrane domains is sufficient to induce -1PRF during the decoding of slippery heptamers. We identify thousands of motifs that potentially trigger -1PRF along with proteomic identifications of 33 predicted human frameshift products. We also identify thousands of splicing-dependent motifs and demonstrate that the splicing-mediated reconfiguration of transmembrane domains alters -1PRF. Finally, we show that most transcripts bearing these motifs are sensitive to the nonsense-mediated decay regulator UPF1, suggesting they modulate mRNA turnover. Our findings show that the misassembly of growing polypeptides can trigger -1PRF, premature termination, and transcript decay.

biochemistry↗

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↗

bioRxiv2023

Ribosomal Frameshifting Selectively Modulates the Biosynthesis, Assembly, and Function 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 ({Delta}F508) 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 {Delta}F508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the {Delta}F508 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 {Delta}F508 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. SignificanceMany diseases stem from imbalances between protein synthesis and degradation that arise from mutations and/ or cellular stressors. The molecular mechanisms responsible for such lapses in cellular proteostasis often coincide with aberrant regulation of protein translation. Here, we identify a structure within the transcript encoding the CFTR chloride channel that allows the ribosome to halt translation in response to its cotranslational misfolding. We show that this motif modifies the assembly, function, and pharmacological properties of the most common cystic fibrosis variant. This crosstalk between the ribosome and nascent polypeptide allows the ribosome to adjust its activity to prevent the synthesis of misfolded proteins. These findings suggest ribosomal frameshifting and premature translational termination plays a fundamental role in protein quality control.

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

Chromenone derivatives as novel pharmacological chaperones for retinitis pigmentosa-linked rod opsin mutants

The correct expression of folded, functional rhodopsin (Rho) is critical for visual perception. However, this seven-transmembrane helical G protein-coupled receptor (GPCR) is prone to mutations with pathological consequences of retinal degeneration in retinitis pigmentosa (RP) due to Rho misfolding. Pharmacological chaperones that stabilize the inherited Rho variants by assisting their folding and membrane targeting could slow the progression of RP. In this study, we employed virtual screening of synthetic compounds with natural product scaffold in conjunction with in vitro and in vivo evaluations to discover a novel chromenone-containing small molecule with favorable pharmacological properties that stabilizes rod opsin. This compound reversibly binds to unliganded bovine rod opsin with an EC50 value comparable to the 9-cis-retinal chromophore analog and partially rescued membrane trafficking of multiple RP-related rod opsin variants in vitro. Importantly, this novel ligand of rod opsin was effective in vivo in murine models, protecting photoreceptors from deterioration caused either by bright light or genetic insult. Together, our current study suggests potential broad therapeutic implications of the new chromenone-containing non-retinoid small molecule against retinal diseases associated with photoreceptor degeneration.

pharmacology and toxicology↗

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↗

bioRxiv2021

Coordination of -1 Programmed Ribosomal Frameshifting by Transcript and Nascent Chain Features Revealed by Deep Mutational Scanning

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. In the alphavirus structural polyprotein, -1PRF is coordinated by a "slippery" sequence in the transcript, an RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4,530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop disrupt -1PRF, mutagenic effects upstream of the slip-site are far more variable. Molecular dynamics simulations of polyprotein biogenesis suggest many of these mutations alter stimulatory forces on the nascent chain through their effects on translocon-mediated cotranslational folding. Finally, we provide evidence suggesting the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

biochemistry↗

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↗