Biology subjects

Kuntz, C. P.

Publications and source records attributed to Kuntz, C. P..

12 recordsLinked to original sources

bioRxiv2026

Protein Stability, Turnover Kinetics, and Abundance Constrain the Scaling of Protein Interaction Networks

The propensity of proteins to form oligomers is ultimately dictated by their structural configuration(s). Proteins that persist in a discrete conformational state may form a limited number of specific interactions while those that sample a broader structural ensemble may instead associate with a wider array of partners. These intrinsic tendencies potentially constrain the way proteins navigate wider interaction networks. In this work, we aggregated and surveyed a wide variety of biophysical, biochemical, and cellular descriptors of the S. cerevisiae proteome to identify biases in the connectivity of its protein-protein interaction network. Using mass spectrometry-based interactome measurements and various protein stability estimates, we find that a disproportionate number of abundant, yet unstable binding proteins act as network hubs. Moreover, we show that these features alone can be used to discriminate between hubs and non-hub proteins with high accuracy (AUROC = 0.898). Interestingly, we find that half-lives of hub proteins depend on whether or not they reside within static complexes and/ or whether they interact with molecular chaperones. Finally, we note that the observed connectivity biases associated with abundant, unstable proteins only pertain to network hubs, but not to the bottlenecks that connect them. Together, our findings reveal how the conformational stability of a protein may constrain its context within protein-protein interaction networks.

systems biology↗

bioRxiv2026

ANYI: The ANnotated Yeast Interactome

Although several existing protein-protein interaction (PPI) databases provide yeast PPI data, none unify large-scale network topology information with detailed biophysical, proteostasis, and regulatory annotations in a single protein-centric framework. To address this gap, we developed the ANnotated Yeast Interactome (ANYI), an open, integrated resource that combines experimental yeast PPIs with sixteen feature annotation types, including protein abundance, half-life, disorder content, post-translational modifications, conformational stability, chaperone interactions, sequence, and structure. ANYI integrates 3,927 proteins with 155 annotation features, forming a unified matrix that enables systematic cross-layer analyses. Available via GitHub and Docker Hub with an interactive network browser for broad accessibility, ANYI provides both experienced and beginner computational scientists with tools to investigate the yeast interactome. For example, users can directly test whether highly connected hub proteins exhibit distinct stability, disorder, or proteostasis signatures relative to peripheral nodes. AVAILABILITY AND IMPLEMENTATIONThe code used to assemble ANYI is available on GitHub at https://github.com/NCEMS/energetic-origins-of-PPI-connectivity and the database itself and interactive browser tool are available on Docker Hub as dannissleypsu/anyi-browser:v1.0.2.

bioinformatics↗

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↗

bioRxiv2025

General Trends in the Calnexin-Dependent Expression and Pharmacological Rescue of Clinical CFTR Variants

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Though most people with CF have one or two copies of the {Delta}F508 mutation, there are hundreds of other distinct CF mutations that vary in their mechanistic effects and response to therapeutics. Endogenous chaperones are known to have divergent effects on the druggability of CF variants. Nevertheless, it remains unclear how this proteostatic modulation is related to the underlying mechanistic effects of distinct classes of CF mutations. Here, we survey the effects of a previously discovered effector (calnexin, CANX) on the expression and pharmacological rescue of 232 CF variants using deep mutational scanning. We find that CANX is generally required for robust plasma membrane expression of the CFTR protein-particularly for CF variants that perturb its second nucleotide binding domain. CANX also appears to be critical for the pharmacological rescue of CF variants with poor basal expression. Though corrector selectivity is generally dictated by the properties of mutations, we find that CANX enhances the sensitivity of CF variants within a domain swapped region of membranes spanning domain 2 to the type III corrector VX-445. Overall, mutagenic trends suggest CANX modulates the later stages of CFTR assembly and disproportionately affects variants bearing mutations within the C-terminal domains. Interestingly, we find that the loss of CANX results in widespread perturbations of CF variant interactomes and that the proteostatic effects of CANX are generally decoupled from changes in CFTR activity. Together, our findings reveal how the proteostasis machinery may shape the variant-specific effects of corrector molecules.

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

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

Towards Generalizable Predictions for the Effects of Mutations on G-Protein Coupled Receptor Expression

Missense mutations that compromise the plasma membrane expression (PME) of integral membrane proteins (MPs) 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 MPs. In this work, we utilize deep mutational scanning data to train a series of artificial neural networks to predict the effects of mutations on the PME of the G-protein coupled receptor (GPCR) rhodopsin from structural and/ or evolutionary features. We show that our best performing network, which we term PMEpred, can differentiate pathogenic rhodopsin variants that induce misfolding from those that primarily compromise signaling. This network also generates statistically significant predictions for the effects of mutations on the PME of another GPCR ({beta}2 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.

biophysics↗

bioRxiv2021

Systematic Profiling of Temperature- and Retinal-Sensitive Rhodopsin Variants by Deep Mutational Scanning

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of "temperature-sensitive" variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and an investigational corrector (9-cis-retinal) on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures is correlated with the response to retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants within this helix appear to disrupt a network of hydrogen bonds that stabilizes a native helical kink. By comparison, mutants that alter a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest this insensitivity primarily arises from an abundance of mutations that enhance its membrane integration, stabilize its native conformation, and/ or perturb the retinal binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings elucidate various factors that mediate the sensitivity of genetic variants to temperature and to small molecule correctors.

biophysics↗

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↗