Biology subjects

Noguera, K.

Publications and source records attributed to Noguera, K..

3 recordsLinked to original sources

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↗

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↗

bioRxiv2020

Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells

Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three proteins implicated in small molecule transport reveals their contribution to increased fitness at low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on timescales and in contexts that enable diverse applications. One Sentence SummaryTo ascertain whether life inevitably finds a way, we continuously evolve an Escherichia coli strain that was not able to escape from engineered biocontainment before, and we find that it does not escape even after 100 days of evolution, nor does it escape when added to mammalian cell culture.

synthetic biology↗