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1.
Cell ; 148(1-2): 150-63, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22265408

RESUMO

The folding and misfolding mechanism of multidomain proteins remains poorly understood. Although thermodynamic instability of the first nucleotide-binding domain (NBD1) of ΔF508 CFTR (cystic fibrosis transmembrane conductance regulator) partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here, we show that ΔF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore ΔF508 CFTR biogenesis. Instead, both ΔF508-NBD1 energetic and the NBD1-MSD2 (membrane-spanning domain 2) interface stabilization are required for wild-type-like folding, processing, and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR-coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of ΔF508 CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multidomain membrane proteins.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Modelos Moleculares , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína
2.
Cell Mol Life Sci ; 81(1): 271, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38888668

RESUMO

Cystic Fibrosis (CF) is a genetic disease caused by mutations in CFTR gene expressing the anion selective channel CFTR located at the plasma membrane of different epithelial cells. The most commonly investigated variant causing CF is F508del. This mutation leads to structural defects in the CFTR protein, which are recognized by the endoplasmic reticulum (ER) quality control system. As a result, the protein is retained in the ER and degraded via the ubiquitin-proteasome pathway. Although blocking ubiquitination to stabilize the CFTR protein has long been considered a potential pharmacological approach in CF, progress in this area has been relatively slow. Currently, no compounds targeting this pathway have entered clinical trials for CF. On the other hand, the emergence of Orkambi initially, and notably the subsequent introduction of Trikafta/Kaftrio, have demonstrated the effectiveness of molecular chaperone-based therapies for patients carrying the F508del variant and even showed efficacy against other variants. These treatments directly target the CFTR variant protein without interfering with cell signaling pathways. This review discusses the limits and potential future of targeting protein ubiquitination in CF.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Ubiquitinação , Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Retículo Endoplasmático/metabolismo , Animais , Mutação , Ubiquitina/metabolismo
3.
Biochem Soc Trans ; 51(3): 1297-1306, 2023 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-37140364

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated anion channel, which is expressed on the apical plasma membrane (PM) of epithelial cells. Mutations in the CFTR gene cause cystic fibrosis (CF), one of the most common genetic diseases among Caucasians. Most CF-associated mutations result in misfolded CFTR proteins that are degraded by the endoplasmic reticulum quality control (ERQC) mechanism. However, the mutant CFTR reaching the PM through therapeutic agents is still ubiquitinated and degraded by the peripheral protein quality control (PeriQC) mechanism, resulting in reduced therapeutic efficacy. Moreover, certain CFTR mutants that can reach the PM under physiological conditions are degraded by PeriQC. Thus, it may be beneficial to counteract the selective ubiquitination in PeriQC to enhance therapeutic outcomes for CF. Recently, the molecular mechanisms of CFTR PeriQC have been revealed, and several ubiquitination mechanisms, including both chaperone-dependent and -independent pathways, have been identified. In this review, we will discuss the latest findings related to CFTR PeriQC and propose potential novel therapeutic strategies for CF.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Chaperonas Moleculares/metabolismo , Mutação
4.
Int J Mol Sci ; 24(3)2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36768629

RESUMO

COPD is a lifestyle-related disease resulting from irreversible damage to respiratory tissues mostly due to chronic exposure to environmental pollutants, including cigarette smoke. Environmental pathogens and pollutants induce the acquired dysfunction of the CFTR Cl- channel, which is invoked in COPD. Despite the increased incidence of CFTR polymorphism R75Q or M470V in COPD patients, the mechanism of how the CFTR variant affects COPD pathogenesis remains unclear. Here, we investigated the impact of CFTR polymorphisms (R75Q, M470V) on the CFTR function in airway epithelial cell models. While wild-type (WT) CFTR suppressed the proinflammatory cytokine production induced by COPD-related pathogens including pyocyanin (PYO), R75Q- or M470V-CFTR failed. Mechanistically, the R75Q- or M470V-CFTR fractional PM activity (FPMA) was significantly lower than WT-CFTR in the presence of PYO. Notably, the CF drug Trikafta corrected the PM expression of R75Q- or M470V-CFTR even upon PYO exposure and consequently suppressed the excessive IL-8 production. These results suggest that R75Q or M470V polymorphism impairs the CFTR function to suppress the excessive proinflammatory response to environmental pathogens associated with COPD. Moreover, Trikafta may be useful to prevent the COPD pathogenesis associated with acquired CFTR dysfunction.


Assuntos
Poluentes Ambientais , Doença Pulmonar Obstrutiva Crônica , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Interleucina-8/genética , Polimorfismo Genético , Exposição Ambiental , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/epidemiologia
5.
J Cell Sci ; 132(3)2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30659120

RESUMO

Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Transporte Biológico , Linhagem Celular , Endocitose/genética , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Transferrina/genética , Transferrina/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética
6.
Mol Cell ; 47(1): 99-110, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22607976

RESUMO

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.


Assuntos
Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Pré-Albumina/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Ácido Azetidinocarboxílico/farmacologia , Proteínas de Ligação ao Cálcio , Retículo Endoplasmático/metabolismo , Glicosilação/efeitos dos fármacos , Células HEK293 , Hexosiltransferases/genética , Humanos , Immunoblotting , Manosidases , Proteínas de Membrana/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Polissacarídeos/metabolismo , Pré-Albumina/química , Pré-Albumina/genética , Estrutura Terciária de Proteína , Desdobramento de Proteína , Interferência de RNA , Via Secretória/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , alfa-Manosidase
7.
Nat Chem Biol ; 9(7): 444-54, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23666117

RESUMO

The most common cystic fibrosis mutation, ΔF508 in nucleotide binding domain 1 (NBD1), impairs cystic fibrosis transmembrane conductance regulator (CFTR)-coupled domain folding, plasma membrane expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and an incompletely understood mechanism, hampering drug development. Given the effect of second-site suppressor mutations, robust ΔF508-CFTR correction most likely requires stabilization of NBD1 energetics and the interface between membrane-spanning domains (MSDs) and NBD1, which are both established primary conformational defects. Here we elucidate the molecular targets of available correctors: class I stabilizes the NBD1-MSD1 and NBD1-MSD2 interfaces, and class II targets NBD2. Only chemical chaperones, surrogates of class III correctors, stabilize human ΔF508-NBD1. Although VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional plasma membrane expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in cystic fibrosis bronchial epithelial cells and intestinal organoids. Thus, the combination of structure-guided correctors represents an effective approach for cystic fibrosis therapy.


Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Animais , Sítios de Ligação , Brônquios/citologia , Membrana Celular/metabolismo , Cricetinae , Fibrose Cística/genética , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Glicosilação , Humanos , Mutação , Nucleotídeos/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química
8.
Front Pharmacol ; 15: 1370676, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38666024

RESUMO

Cystic fibrosis (CF) is a monogenetic disease caused by the mutation of CFTR, a cAMP-regulated Cl- channel expressing at the apical plasma membrane (PM) of epithelia. ∆F508-CFTR, the most common mutant in CF, fails to reach the PM due to its misfolding and premature degradation at the endoplasmic reticulum (ER). Recently, CFTR modulators have been developed to correct CFTR abnormalities, with some being used as therapeutic agents for CF treatment. One notable example is Trikafta, a triple combination of CFTR modulators (TEZ/ELX/IVA), which significantly enhances the functionality of ΔF508-CFTR on the PM. However, there's room for improvement in its therapeutic effectiveness since TEZ/ELX/IVA doesn't fully stabilize ΔF508-CFTR on the PM. To discover new CFTR modulators, we conducted a virtual screening of approximately 4.3 million compounds based on the chemical structures of existing CFTR modulators. This effort led us to identify a novel CFTR ligand named FR3. Unlike clinically available CFTR modulators, FR3 appears to operate through a distinct mechanism of action. FR3 enhances the functional expression of ΔF508-CFTR on the apical PM in airway epithelial cell lines by stabilizing NBD1. Notably, FR3 counteracted the degradation of mature ΔF508-CFTR, which still occurs despite the presence of TEZ/ELX/IVA. Furthermore, FR3 corrected the defective PM expression of a misfolded ABCB1 mutant. Therefore, FR3 may be a potential lead compound for addressing diseases resulting from the misfolding of ABC transporters.

9.
J Cell Biol ; 223(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38722278

RESUMO

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana , Ubiquitina-Proteína Ligases , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Ligação a DNA , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
10.
Cells ; 12(23)2023 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-38067172

RESUMO

The ubiquitin E3 ligase UBE3C promotes the proteasomal degradation of cytosolic proteins and endoplasmic reticulum (ER) membrane proteins. UBE3C is proposed to function downstream of the RNF185/MBRL ER-associated degradation (ERAD) branch, contributing to the ERAD of select membrane proteins. Here, we report that UBE3C facilitates the ERAD of misfolded CFTR, even in the absence of both RNF185 and its functional ortholog RNF5 (RNF5/185). Unlike RNF5/185, UBE3C had a limited impact on the ubiquitination of misfolded CFTR. UBE3C knockdown (KD) resulted in an additional increase in the functional ∆F508-CFTR channels on the plasma membrane when combined with the RNF5/185 ablation, particularly in the presence of clinically used CFTR modulators. Interestingly, although UBE3C KD failed to attenuate the ERAD of insig-1, it reduced the ERAD of misfolded ∆Y490-ABCB1 and increased cell surface expression. UBE3C KD also stabilized the mature form of ∆F508-CFTR and increased the cell surface level of T70-CFTR, a class VI CFTR mutant. These results suggest that UBE3C plays a vital role in the ERAD of misfolded CFTR and ABCB1, even within the RNF5/185-independent ERAD pathway, and it may also be involved in maintaining the peripheral quality control of CFTR.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Ubiquitina-Proteína Ligases , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Degradação Associada com o Retículo Endoplasmático , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Dobramento de Proteína
11.
Biochem Pharmacol ; 215: 115730, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37543348

RESUMO

The E3 ubiquitin ligase RFFL is an apoptotic inhibitor highly expressed in cancers and its knockdown suppresses cancer cell growth and sensitizes to chemotherapy. RFFL also participates in peripheral protein quality control which removes the functional cell surface ΔF508-CFTR channel and reduces the efficacy of pharmaceutical therapy for cystic fibrosis (CF). Although RFFL inhibitors have therapeutic potential for both cancer and CF, they remain undiscovered. Here, a chemical array screening has identified α-tocopherol succinate (αTOS) as an RFFL ligand. NMR analysis revealed that αTOS directly binds to RFFL's substrate-binding region without affecting the E3 enzymatic activity. Consequently, αTOS inhibits the RFFL-substrate interaction, ΔF508-CFTR ubiquitination and elimination from the plasma membrane of epithelial cells, resulting in the increased functional CFTR channel. Among the α-tocopherol (αTOL) analogs we tested, only αTOS inhibited the RFFL-substrate interaction and increased the cell surface ΔF508-CFTR, depending on RFFL expression. Similarly, the unique proapoptotic effect of αTOS was dependent on RFFL expression. Thus, unlike other αTOL analogs, αTOS acts as an RFFL protein-protein interaction inhibitor which may explain its unique biological properties among αTOL analogs. Moreover, αTOS may act as a CFTR stabilizer, a novel class of drugs that extend cell surface ΔF508-CFTR lifetime.


Assuntos
Fibrose Cística , alfa-Tocoferol , Humanos , alfa-Tocoferol/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Antioxidantes/farmacologia , Fibrose Cística/tratamento farmacológico , Apoptose
12.
Nat Chem Biol ; 6(1): 25-33, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19966789

RESUMO

Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Histona Desacetilases/metabolismo , Mutação , Animais , Brônquios/metabolismo , Membrana Celular/metabolismo , Cricetinae , Células Epiteliais/metabolismo , Inativação Gênica , Humanos , Ácidos Hidroxâmicos/química , Desnaturação Proteica , Dobramento de Proteína , RNA Interferente Pequeno/metabolismo , Vorinostat
13.
Front Mol Biosci ; 9: 840649, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35355508

RESUMO

The peripheral protein quality control (periQC) system eliminates the conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR), including ∆F508-CFTR, from the plasma membrane (PM) and limits the efficacy of pharmacological therapy for cystic fibrosis (CF). The ubiquitin (Ub) ligase RFFL is responsible for the chaperone-independent ubiquitination and lysosomal degradation of CFTR in the periQC. Here, we report that the Ub ligase RNF34 participates in the CFTR periQC in parallel to RFFL. An in vitro study reveals that RNF34 directly recognizes the CFTR NBD1 and selectively promotes the ubiquitination of unfolded proteins. RNF34 was localized in the cytoplasm and endosomes, where RFFL was equally colocalized. RNF34 ablation increased the PM density as well as the mature form of ∆F508-CFTR rescued at low temperatures. RFFL ablation, with the exception of RNF34 ablation, increased the functional PM expression of ∆F508-CFTR upon a triple combination of CFTR modulators (Trikafta) treatment by inhibiting the K63-linked polyubiquitination. Interestingly, simultaneous ablation of RNF34 and RFFL dramatically increased the functional PM ∆F508-CFTR by inhibiting the ubiquitination in the post-Golgi compartments. The CFTR-NLuc assay demonstrates that simultaneous ablation of RNF34 and RFFL dramatically inhibits the degradation of mature ∆F508-CFTR after Trikafta treatment. Therefore, these results suggest that RNF34 plays a crucial role in the CFTR periQC, especially when there is insufficient RFFL. We propose that simultaneous inhibition of RFFL and RNF34 may improve the efficacy of CFTR modulators.

14.
J Biochem ; 170(4): 453-461, 2021 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33982090

RESUMO

Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Biotina/farmacologia , Mapeamento de Interação de Proteínas/métodos , Animais , Biotina/administração & dosagem , Biotinilação/métodos , Encéfalo/metabolismo , Proteínas CLOCK/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Dieta/métodos , Fibroblastos/metabolismo , Genótipo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Músculos/metabolismo , Coloração e Rotulagem/métodos
15.
Pharmaceuticals (Basel) ; 13(4)2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32331485

RESUMO

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene decrease the structural stability and function of the CFTR protein, resulting in cystic fibrosis. Recently, the effect of CFTR-targeting combination therapy has dramatically increased, and it is expected that add-on drugs that modulate the CFTR surrounding environment will further enhance their effectiveness. Various interacting proteins have been implicated in the structural stability of CFTR and, among them, molecules involved in CFTR ubiquitylation are promising therapeutic targets as regulators of CFTR degradation. This review focuses on the ubiquitylation mechanism that contributes to the stability of mutant CFTR at the endoplasmic reticulum (ER) and post-ER compartments and discusses the possibility as a pharmacological target for cystic fibrosis (CF).

16.
Biochim Biophys Acta ; 1783(1): 153-62, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17936375

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) is transported to the plasma membrane from endoplasmic reticulum (ER) through the Golgi. Crucial to these trafficking events is the role of not only the proteinous factors but also the membrane lipids. However, the involvement of lipids, such as phospholipids, on the regulation of CFTR trafficking has been largely unexplored. Here, we show that the inhibition of phospholipase D (PLD)-mediated phosphatidic acid (PA) formation by 1-butanol inhibited the maturation and export of CFTR from the ER. Exogenously added PA reversed these effects. Moreover, knock down of PLD1 by small interfering RNA decreased the expression of mature CFTR. Interestingly, sustaining the level of PA, by the addition of excess PA in the presence of PA phosphatase inhibitor, attenuated the transport of CFTR from the Golgi to plasma membrane and the retrograde transport of DeltaF508 CFTR to the cytoplasm, a necessary step for the ER-associated degradation of DeltaF508 CFTR. These results indicated that the metabolism of PA modulated the intracellular dynamics and trafficking of CFTR.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Linhagem Celular , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Retículo Endoplasmático/metabolismo , Deleção de Genes , Complexo de Golgi/metabolismo , Fosfatidato Fosfatase/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico
17.
Biochim Biophys Acta ; 1783(9): 1585-94, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18457676

RESUMO

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.


Assuntos
Calnexina/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Calnexina/genética , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/química , Epitélio/fisiologia , Camundongos , Camundongos Knockout , Dobramento de Proteína , Deleção de Sequência
19.
Bio Protoc ; 9(22): e3430, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33654926

RESUMO

Ubiquitylation is a common post-translational modification of cellular proteins that results in proteasomal and lysosomal degradations. Ubiquitylation is generally measured by methods such as immunoblotting using anti-ubiquitin antibodies after isolating the protein-of-interest by denaturing immunoprecipitation. The following protocol can be used to easily quantify the ubiquitylation of the protein-of-interest tagged with biotin by ELISA.

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