Structural and Mechanistic Principles of ABC Transporters

Annual Review of Biochemistry, Год журнала: 2020, Номер 89(1), С. 605 - 636

Опубликована: Июнь 20, 2020

ATP-binding cassette (ABC) transporters constitute one of the largest and most ancient protein superfamilies found in all living organisms. They function as molecular machines by coupling ATP binding, hydrolysis, phosphate release to translocation diverse substrates across membranes. The range from vitamins, steroids, lipids, ions peptides, proteins, polysaccharides, xenobiotics. ABC undergo substantial conformational changes during substrate translocation. A comprehensive understanding their inner workings thus requires linking these structural rearrangements different functional state transitions. Recent advances single-particle cryogenic electron microscopy have not only delivered crucial information on architecture several medically relevant supramolecular assemblies, including ATP-sensitive potassium channel peptide-loading complex, but also made it possible explore entire space nanomachines under turnover conditions thereby gain detailed mechanistic insights into mode action.

Язык: Английский

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Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing

Biologics, Год журнала: 2021, Номер Volume 15, С. 353 - 361

Опубликована: Авг. 1, 2021

Abstract: Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, accurate method of genome editing tool in all living cells utilized many applied disciplines. Guide RNA (gRNA) CRISPR-associated proteins are two essential components CRISPR/Cas-9 system. The mechanism contains three steps, recognition, cleavage, repair. designed sgRNA recognizes target sequence gene interest through a complementary base pair. While Cas-9 nuclease makes double-stranded breaks at site 3 pair upstream to protospacer adjacent motif, then break repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. genome-editing has wide number applications areas including medicine, agriculture, biotechnology. In it could help design new grains improve nutritional value. being investigated for cancers, HIV, therapy such as sickle cell disease, cystic fibrosis, Duchenne muscular dystrophy. technology also regulation specific genes advanced modification protein. However, immunogenicity, effective delivery systems, off-target effect, ethical issues have been major barriers extend clinical applications. Although becomes era molecular biology countless roles ranging from basic researches applications, there still challenges rub practical various improvements needed overcome obstacles. Keywords: CRISPR, Cas-9, sgRNA, gene-editing, mechanism,

Язык: Английский

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Mechanism of CFTR correction by type I folding correctors

Cell, Год журнала: 2022, Номер 185(1), С. 158 - 168.e11

Опубликована: Янв. 1, 2022

Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects ΔF508 mutant and widely used to treat patients. To investigate molecular mechanism their action, we determined cryo-electron microscopy structures in complex with FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket first transmembrane domain (TMD1), linking together four helices that thermodynamically unstable. Mutating residues at binding site rendered ΔF508-CFTR insensitive tezacaftor, underscoring functional significance structural discovery. results support stabilize TMD1 an early stage biogenesis, prevent its premature degradation, thereby allosterically rescuing many disease-causing mutations.

Язык: Английский

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Molecular structures reveal synergistic rescue of Δ508 CFTR by Trikafta modulators

Science, Год журнала: 2022, Номер 378(6617), С. 284 - 290

Опубликована: Окт. 20, 2022

The predominant mutation causing cystic fibrosis, a deletion of phenylalanine 508 (Δ508) in the fibrosis transmembrane conductance regulator (CFTR), leads to severe defects CFTR biogenesis and function. advanced therapy Trikafta combines folding corrector tezacaftor (VX-661), channel potentiator ivacaftor (VX-770), dual-function modulator elexacaftor (VX-445). However, it is unclear how exerts its effects, part because structure Δ508 unknown. Here, we present cryo-electron microscopy structures absence presence modulators. When used alone, partially rectified interdomain assembly CFTR, but when combined with type I corrector, did so fully. These data illustrate different modulators synergistically rescue

Язык: Английский

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Ins and outs of AlphaFold2 transmembrane protein structure predictions

Cellular and Molecular Life Sciences, Год журнала: 2022, Номер 79(1)

Опубликована: Янв. 1, 2022

Transmembrane (TM) proteins are major drug targets, but their structure determination, a prerequisite for rational design, remains challenging. Recently, the DeepMind's AlphaFold2 machine learning method greatly expanded structural coverage of sequences with high accuracy. Since employed algorithm did not take specific properties TM into account, reliability generated structures should be assessed. Therefore, we quantitatively investigated quality at genome scales, level ABC protein superfamily folds and membrane (e.g. dimer modeling stability in molecular dynamics simulations). We tested template-free prediction challenging CASP14 target several published after training. Our results suggest that performs well case its neural network is overfitted. conclude cautious applications models will advance protein-associated studies an unexpected level.

Язык: Английский

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CFTR function, pathology and pharmacology at single-molecule resolution

Nature, Год журнала: 2023, Номер 616(7957), С. 606 - 614

Опубликована: Март 22, 2023

Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes 1 . Alterations in CFTR cause fibrosis, a fatal disease without cure 2,3 Electrophysiological properties of have been analysed for decades 4–6 structure CFTR, determined two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology kinetic simulations to show nucleotide-binding domains (NBDs) human dimerize before opening. exhibits allosteric gating mechanism which conformational changes within NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. potentiators ivacaftor GLPG1837 enhance activity increasing pore opening while NBDs dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) ATPase site both reduce efficiency NBD dimerization. These findings collectively enable framing informs on search more efficacious clinical therapies.

Язык: Английский

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Analysis of AlphaMissense data in different protein groups and structural context

Scientific Data, Год журнала: 2024, Номер 11(1)

Опубликована: Май 14, 2024

Abstract Single amino acid substitutions can profoundly affect protein folding, dynamics, and function. The ability to discern between benign pathogenic is pivotal for therapeutic interventions research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor pathogenicity missense variants. Since heterogenous performance on different types proteins be expected, we assessed efficacy across several groups (e.g. soluble, transmembrane, mitochondrial proteins) regions intramembrane, membrane interacting, high confidence AlphaFold segments) using ClinVar data validation. Our comprehensive evaluation showed that delivers outstanding performance, with MCC scores predominantly 0.6 0.74. We observed low disordered datasets related CFTR ABC protein. However, superior was shown when benchmarked against quality CFTR2 database. results emphasizes AlphaMissense’s potential pinpointing functional hot spots, its likely surpassing benchmarks calculated from ProteinGym datasets.

Язык: Английский

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TRPV4: A Physio and Pathophysiologically Significant Ion Channel

International Journal of Molecular Sciences, Год журнала: 2020, Номер 21(11), С. 3837 - 3837

Опубликована: Май 28, 2020

Transient Receptor Potential (TRP) channels are a family of ion whose members distributed among all kinds animals, from invertebrates to vertebrates. The importance these molecules is exemplified by the variety physiological roles they play. Perhaps, most extensively studied member this TRPV1 channel; nonetheless, activity TRPV4 has been associated several physio and pathophysiological processes, its dysfunction can lead severe consequences. Several lines evidence derived animal models even clinical trials in humans highlight as therapeutic target protein that will receive more attention near future, be reviewed here.

Язык: Английский

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Progression of Cystic Fibrosis Lung Disease from Childhood to Adulthood: Neutrophils, Neutrophil Extracellular Trap (NET) Formation, and NET Degradation

Genes, Год журнала: 2019, Номер 10(3), С. 183 - 183

Опубликована: Фев. 26, 2019

Genetic defects in cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene cause CF. Infants with CFTR mutations show a peribronchial neutrophil infiltration prior to the establishment of infection their lung. The inflammatory response progressively increases children that include both upper and lower airways. Infectious leads an increase mucus viscosity plugging small medium-size bronchioles. Eventually, neutrophils chronically infiltrate airways biofilm or chronic bacterial infection. Perpetual airway inflammation destroy lungs, which increased morbidity eventual mortality most patients Studies have now established cytotoxins, extracellular DNA, traps (NETs) are associated clogging lung injury In addition opportunistic pathogens, various aspects CF milieux (e.g., pH, salt concentration, phenotypes) influence NETotic capacity neutrophils. milieu may promote survival pro-inflammatory aberrant NETosis, rather than anti-inflammatory apoptotic death these cells. Degrading NETs helps manage disease; since DNAse treatment release cytotoxins from NETs, further improvements needed degrade maximal positive effects. Neutrophil-T cell interactions be important regulating viral infection-mediated pulmonary exacerbations infections. Therefore, clarifying role disease identifying therapies preserve effects neutrophils, while reducing detrimental cytotoxic components, essential achieving innovative therapeutic advances.

Язык: Английский

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Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

eLife, Год журнала: 2020, Номер 9

Опубликована: Май 27, 2020

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety chemotherapeutic agents and native substrates. Previously, MRP1 were determined in inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track conformational changes bovine (bMRP1) real time. We also structure bMRP1 under active turnover conditions. Our results show substrate stimulates ATP hydrolysis by accelerating IF-to-OF transition. The rate-limiting step cycle dissociation nucleotide-binding-domain dimer, while per se does not reset resting state. combination structural kinetic data illustrates how different conformations temporally linked alter dynamics achieve transport.

Язык: Английский

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