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1.
Int J Mol Sci ; 24(21)2023 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-37958724

RESUMO

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a large multi-spanning membrane protein that is susceptible to misfolding and aggregation. We have identified here the region responsible for this instability. Temperature-induced aggregation of C-terminally truncated versions of CFTR demonstrated that all truncations up to the second transmembrane domain (TMD2), including the R region, largely resisted aggregation. Limited proteolysis identified a folded structure that was prone to aggregation and consisted of TMD2 and at least part of the Regulatory Region R. Only when both TM7 (TransMembrane helix 7) and TM8 were present, TMD2 fragments became as aggregation-sensitive as wild-type CFTR, in line with increased thermo-instability of late CFTR nascent chains and in silico prediction of aggregation propensity. In accord, isolated TMD2 was degraded faster in cells than isolated TMD1. We conclude that TMD2 extended at its N-terminus with part of the R region forms a protease-resistant structure that induces heat instability in CFTR and may be responsible for its limited intracellular stability.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Temperatura Alta , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Membrana Celular/metabolismo , Proteólise , Temperatura
2.
PLoS Biol ; 15(5): e2000779, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28510592

RESUMO

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , RNA de Transferência/metabolismo , Mutação Silenciosa , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células HEK293 , Células HeLa , Humanos , Polimorfismo de Nucleotídeo Único , Estabilidade Proteica , Relação Estrutura-Atividade
3.
Nat Chem Biol ; 9(11): 731-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24077179

RESUMO

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.


Assuntos
Di-Hidropiridinas/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Via Secretória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Di-Hidropiridinas/química , Retículo Endoplasmático/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Estrutura Molecular , Receptores Notch/metabolismo , Relação Estrutura-Atividade , Peixe-Zebra/metabolismo
4.
Curr Opin Cell Biol ; 16(4): 343-9, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15261665

RESUMO

The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. ER quality control guided by these chaperones is essential for life. Whereas correctly folded proteins are exported from the ER, misfolded proteins are retained and selectively degraded. At least two main chaperone classes, BiP and calnexin/calreticulin, are active in ER quality control. Folding factors usually are found in complexes. Recent work emphasises more than ever that chaperones act in concert with co-factors and with each other.


Assuntos
Retículo Endoplasmático/metabolismo , Dobramento de Proteína , Animais , Calnexina/metabolismo , Calreticulina/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Lectinas/metabolismo , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional
5.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34292306

RESUMO

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Presenilina-1/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Animais , Transporte Biológico , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Linhagem Celular , Linhagem Celular Tumoral , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Endopeptidases/química , Endopeptidases/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Neurônios/citologia , Presenilina-1/química , Presenilina-1/genética , Cultura Primária de Células , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Ratos , Ratos Wistar , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
6.
J Mol Biol ; 433(13): 166955, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33771570

RESUMO

ABC transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: (i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and (ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citosol/metabolismo , Biossíntese de Proteínas , Dobramento de Proteína , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Evolução Molecular , Genes Supressores , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Domínios Proteicos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína
7.
J Cyst Fibros ; 19 Suppl 1: S19-S24, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31759907

RESUMO

Structural biology and functional studies are a powerful combination to elucidate fundamental knowledge about the cystic fibrosis transmembrane conductance regulator (CFTR). Here, we discuss the latest findings, including how clinically-approved drugs restore function to mutant CFTR, leading to better clinical outcomes for people with cystic fibrosis (CF). Despite the prospect of regulatory approval of a CFTR-targeting therapy for most CF mutations, strenuous efforts are still needed to fully comprehend CFTR structure-and-function for the development of better drugs to enable people with CF to live full and active lives.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística , Terapia de Alvo Molecular/métodos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Mutação , Resultado do Tratamento
8.
J Vis Exp ; (144)2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30829321

RESUMO

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (<30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.


Assuntos
Dobramento de Proteína , Poluentes Radioativos/efeitos adversos , Movimento Celular , Transfecção
9.
Life Sci Alliance ; 2(1)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30659068

RESUMO

Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/patologia , Dobramento de Proteína/efeitos dos fármacos , Alelos , Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Biópsia , Regulador de Condutância Transmembrana em Fibrose Cística/classificação , Genótipo , Células HEK293 , Humanos , Mutação , Organoides/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Quinolonas/farmacologia , Reto/patologia , Transfecção
10.
Curr Opin Pharmacol ; 34: 83-90, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-29055231

RESUMO

Pharmacological intervention to treat the lethal genetic disease cystic fibrosis has become reality, even for the severe, most common folding mutant F508del CFTR. CFTR defects range from absence of the protein, misfolding that leads to degradation rather than cell-surface localization (such as F508del), to functional chloride-channel defects on the cell surface. Corrector and potentiator drugs improve cell-surface location and channel activity, respectively, and combination therapy of two correctors and a potentiator have shown synergy. Several combinations are in the drug-development pipeline and although the primary defect is not repaired, rescue levels are reaching those resembling a cure for CF. Combination therapy with correctors may also improve functional CFTR mutants and benefit patients on potentiator therapy.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Humanos , Dobramento de Proteína
11.
PLoS One ; 5(11): e15458, 2010 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21152102

RESUMO

In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mutação , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Fibrose Cística/genética , Fibrose Cística/terapia , Teste de Complementação Genética , Terapia Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Biossíntese de Proteínas , Homologia de Sequência de Aminoácidos
13.
J Cell Biol ; 179(5): 951-63, 2007 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-18056412

RESUMO

Gamma-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by gamma-secretase produces a fragment, Abeta(42), that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Abeta processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of gamma-secretase. Here, we provide direct evidence that the gamma-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)-linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.


Assuntos
Secretases da Proteína Precursora do Amiloide/biossíntese , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Células CHO , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Linhagem Celular , Cricetinae , Cricetulus , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Metilaminas/farmacologia , Proteínas Mutantes/metabolismo , Presenilina-1/biossíntese , Transporte Proteico/efeitos dos fármacos , Ratos , Proteínas de Transporte Vesicular/metabolismo
14.
Mol Cell ; 20(2): 277-87, 2005 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-16246729

RESUMO

The folding process for newly synthesized, multispanning membrane proteins in the endoplasmic reticulum (ER) is largely unknown. Here, we describe early folding events of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC-transporter family. In vitro translation of CFTR in the presence of semipermeabilized cells allowed us to investigate this protein during nascent chain elongation. We found that CFTR folds mostly during synthesis as determined by protease susceptibility. C-terminally truncated constructs showed that individual CFTR domains formed well-defined structures independent of C-terminal parts. We conclude that the multidomain protein CFTR folds mostly cotranslationally, domain by domain.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dobramento de Proteína , Linhagem Celular Tumoral , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/química , Retículo Endoplasmático/metabolismo , Humanos , Técnicas In Vitro , Modelos Biológicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo
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