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1.
Nat Commun ; 11(1): 4258, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848127

RESUMO

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação , Substituição de Aminoácidos , Sítios de Ligação/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células HEK293 , Humanos , Técnicas In Vitro , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Dobramento de Proteína , Modificação Traducional de Proteínas/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Supressão Genética , Temperatura
2.
Nat Commun ; 11(1): 3922, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764664

RESUMO

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT's native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico Ativo , Resistência a Medicamentos/genética , Feminino , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligopeptídeos/metabolismo , Oócitos/metabolismo , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/genética , Xenopus laevis
3.
Nature ; 585(7824): 251-255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848248

RESUMO

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas Relacionadas à Autofagia/química , Proteína C9orf72/química , Proteína C9orf72/genética , Proteínas de Transporte/química , Microscopia Crioeletrônica , Demência Frontotemporal/genética , Haploinsuficiência , Complexos Multiproteicos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Demência Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Domínios Proteicos
4.
Nat Commun ; 11(1): 3751, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719344

RESUMO

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.


Assuntos
Ribonucleoproteínas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Rotação , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação/genética , Domínios Proteicos , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Subunidades Ribossômicas Maiores/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
5.
Nat Commun ; 11(1): 2687, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483116

RESUMO

Injury of corpus cavernosa results in erectile dysfunction, but its treatment has been very difficult. Here we construct heparin-coated 3D-printed hydrogel scaffolds seeded with hypoxia inducible factor-1α (HIF-1α)-mutated muscle-derived stem cells (MDSCs) to develop bioengineered vascularized corpora. HIF-1α-mutated MDSCs significantly secrete various angiogenic factors in MDSCs regardless of hypoxia or normoxia. The biodegradable scaffolds, along with MDSCs, are implanted into corpus cavernosa defects in a rabbit model to show good histocompatibility with no immunological rejection, support vascularized tissue ingrowth, and promote neovascularisation to repair the defects. Evaluation of morphology, intracavernosal pressure, elasticity and shrinkage of repaired cavernous tissue prove that the bioengineered corpora scaffolds repair the defects and recover penile erectile and ejaculation function successfully. The function recovery restores the reproductive capability of the injured male rabbits. Our work demonstrates that the 3D-printed hydrogels with angiogenic cells hold great promise for penile reconstruction to restore reproductive capability of males.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Pênis/lesões , Transplante de Células-Tronco/métodos , Animais , Sobrevivência Celular , Modelos Animais de Doenças , Disfunção Erétil/diagnóstico por imagem , Disfunção Erétil/fisiopatologia , Disfunção Erétil/cirurgia , Feminino , Heparina , Humanos , Hidrogéis , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos Nus , Proteínas Mutantes/genética , Neovascularização Fisiológica , Pênis/irrigação sanguínea , Pênis/fisiopatologia , Gravidez , Impressão Tridimensional , Coelhos , Tecidos Suporte , Transfecção
6.
PLoS One ; 15(6): e0234375, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555682

RESUMO

Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RDB mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RDB kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.


Assuntos
Rim/anormalidades , Rim/efeitos dos fármacos , Quercetina/farmacologia , beta Catenina/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Vimentina/metabolismo , beta Catenina/química , beta Catenina/genética
7.
PLoS One ; 15(6): e0234394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574176

RESUMO

In the BACHD mouse model of Huntington's disease (HD), deletion of the N17 domain of the Huntingtin gene (BACHDΔN17, Q97) has been reported to lead to nuclear accumulation of mHTT and exacerbation of motor deficits, neuroinflammation and striatal atrophy (Gu et al., 2015). Here we characterized the effect of N17 deletion on dorsolateral striatal medium spiny neurons (MSNs) in BACHDΔN17 (Q97) and BACWTΔN17 (Q31) mice by comparing them to MSNs in wildtype (WT) mice. Mice were characterized on a series of motor tasks and subsequently whole cell patch clamp recordings with simultaneous biocytin filling of MSNs in in vitro striatal slices from these mice were used to comprehensively assess their physiological and morphological features. Key findings include that: Q97 mice exhibit impaired gait and righting reflexes but normal tail suspension reflexes and normal coats while Q31 mice do not differ from WT; intrinsic membrane and action potential properties are altered -but differentially so- in MSNs from Q97 and from Q31 mice; excitatory and inhibitory synaptic currents exhibit higher amplitudes in Q31 but not Q97 MSNs, while excitatory synaptic currents occur at lower frequency in Q97 than in WT and Q31 MSNs; there is a reduced total dendritic length in Q31 -but not Q97- MSNs compared to WT, while spine density and number did not differ in MSNs in the three groups. The findings that Q31 MSNs differed from Q97 and WT neurons with regard to some physiological features and structurally suggest a novel role of the N17 domain in the function of WT Htt. The motor phenotype seen in Q97 mice was less robust than that reported in an earlier study (Gu et al., 2015), and the alterations to MSN physiological properties were largely consistent with changes reported previously in a number of other mouse models of HD. Together this study indicates that N17 plays a role in the modulation of the properties of MSNs in both mHtt and WT-Htt mice, but does not markedly exacerbate HD-like pathogenesis in the BACHD model.


Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , Potenciais de Ação , Animais , Corpo Estriado/patologia , Corpo Estriado/fisiopatologia , Dendritos/patologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Feminino , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/fisiologia , Doença de Huntington/patologia , Doença de Huntington/fisiopatologia , Coxeadura Animal/genética , Coxeadura Animal/fisiopatologia , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Neurônios/patologia , Neurônios/fisiologia , Domínios Proteicos , Reflexo Anormal/genética , Reflexo Anormal/fisiologia , Deleção de Sequência
8.
PLoS One ; 15(5): e0233017, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407401

RESUMO

Myotonia congenita and hypokalemic periodic paralysis type 2 are both rare genetic channelopathies caused by mutations in the CLCN1 gene encoding voltage-gated chloride channel CLC-1 and the SCN4A gene encoding voltage-gated sodium channel Nav1.4. The patients with concomitant mutations in both genes manifested different unique symptoms from mutations in these genes separately. Here, we describe a patient with myotonia and periodic paralysis in a consanguineous marriage pedigree. By using whole-exome sequencing, a novel F306S variant in the CLCN1 gene and a known R222W mutation in the SCN4A gene were identified in the pedigree. Patch clamp analysis revealed that the F306S mutant reduced the opening probability of CLC-1 and chloride conductance. Our study expanded the CLCN1 mutation database. We emphasized the value of whole-exome sequencing for differential diagnosis in atypical myotonic patients.


Assuntos
Canais de Cloreto/genética , Paralisia Periódica Hipopotassêmica/complicações , Paralisia Periódica Hipopotassêmica/genética , Miotonia Congênita/complicações , Miotonia Congênita/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , China , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Consanguinidade , Sequência Conservada , Diagnóstico Diferencial , Feminino , Células HEK293 , Humanos , Paralisia Periódica Hipopotassêmica/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Miotonia Congênita/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Linhagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequenciamento Completo do Exoma , Adulto Jovem
9.
Nat Commun ; 11(1): 2643, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457390

RESUMO

Amyloid aggregation of α-synuclein (α-syn) is closely associated with Parkinson's disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of α-syn have been identified causative to the early onset of familial PD. Here, we report the cryo-EM structure of an α-syn fibril formed by N-terminally acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a distinct fold of α-syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) α-syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with an enhanced seeding capability than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of α-syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs.


Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , Acetilação , Sequência de Aminoácidos , Substituição de Aminoácidos , Amiloide/química , Amiloide/genética , Amiloide/ultraestrutura , Microscopia Crioeletrônica , Humanos , Microscopia de Força Atômica , Modelos Moleculares , Proteínas Mutantes/ultraestrutura , Mutação de Sentido Incorreto , Conformação Proteica , Estabilidade Proteica , Eletricidade Estática , alfa-Sinucleína/ultraestrutura
10.
Nucleic Acids Res ; 48(11): 6068-6080, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374842

RESUMO

We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Quinases/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/efeitos da radiação , Apoptose/efeitos da radiação , Dano ao DNA/efeitos da radiação , Fluorescência , Genes Dominantes , Genes Reporter , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos da radiação , Proteínas Quinases/genética , Transcrição Genética/efeitos da radiação , Raios Ultravioleta
11.
PLoS One ; 15(5): e0233439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469934

RESUMO

In epithelial cells, the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl- channel, plays a key role in water and electrolytes secretion. A dysfunctional CFTR leads to the dehydration of the external environment of the cells and to the production of viscous mucus in the airways of cystic fibrosis patients. Here, we applied the quadriwave lateral shearing interferometry (QWLSI), a quantitative phase imaging technique based on the measurement of the light wave shift when passing through a living sample, to study water transport regulation in human airway epithelial CFBE and CHO cells expressing wild-type, G551D- and F508del-CFTR. We were able to detect phase variations during osmotic challenges and confirmed that cellular volume changes reflecting water fluxes can be detected with QWLSI. Forskolin stimulation activated a phase increase in all CFBE and CHO cell types. This phase variation was due to cellular volume decrease and intracellular refractive index increase and was completely blocked by mercury, suggesting an activation of a cAMP-dependent water efflux mediated by an endogenous aquaporin (AQP). AQP3 mRNAs, not AQP1, AQP4 and AQP5 mRNAs, were detected by RT-PCR in CFBE cells. Readdressing the F508del-CFTR protein to the cell surface with VX-809 increased the detected water efflux in CHO but not in CFBE cells. However, VX-770, a potentiator of CFTR function, failed to further increase the water flux in either G551D-CFTR or VX-809-corrected F508del-CFTR expressing cells. Our results show that QWLSI could be a suitable technique to study water transport in living cells. We identified a CFTR and cAMP-dependent, mercury-sensitive water transport in airway epithelial and CHO cells that might be due to AQP3. This water transport appears to be affected when CFTR is mutated and independent of the chloride channel function of CFTR.


Assuntos
Aquaporina 3/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Respiratória/metabolismo , Água/metabolismo , Aminofenóis/farmacologia , Animais , Aquaporina 3/genética , Transporte Biológico Ativo/efeitos dos fármacos , Fenômenos Biofísicos , Brônquios/citologia , Brônquios/metabolismo , Células CHO , Linhagem Celular , Agonistas dos Canais de Cloreto/farmacologia , Colforsina/farmacologia , Cricetulus , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Humanos , Microscopia de Interferência , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Osmose , Quinolonas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mucosa Respiratória/citologia
13.
Hum Genet ; 139(5): 657-673, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32219518

RESUMO

GM1-gangliosidosis, a lysosomal storage disorder, is associated with ~ 161 missense variants in the GLB1 gene. Affected patients present with ß-galactosidase (ß-Gal) deficiency in lysosomes. Loss of function in ER-retained misfolded enzymes with missense variants is often due to subcellular mislocalization. Deoxygalactonojirimycin (DGJ) and its derivatives are pharmaceutical chaperones that directly bind to mutated ß-Gal in the ER promoting its folding and trafficking to lysosomes and thus enhancing its activity. An Emirati child has been diagnosed with infantile GM1-gangliosidosis carrying the reported p.D151Y variant. We show that p.D151Y ß-Gal in patient's fibroblasts retained < 1% residual activity due to impaired processing and trafficking. The amino acid substitution significantly affected the enzyme conformation; however, p.D151Y ß-Gal was amenable for partial rescue in the presence of glycerol or at reduced temperature where activity was enhanced with ~ 2.3 and 7 folds, respectively. The butyl (NB-DGJ) and nonyl (NN-DGJ) derivatives of DGJ chaperoning function were evaluated by measuring their IC50s and ability to stabilize the wild-type ß-Gal against thermal degradation. Although NN-DGJ showed higher affinity to ß-Gal, it did not show a significant enhancement in p.D151Y ß-Gal activity. However, NB-DGJ promoted p.D151Y ß-Gal maturation and enhanced its activity up to ~ 4.5% of control activity within 24 h which was significantly increased to ~ 10% within 6 days. NB-DGJ enhancement effect was sustained over 3 days after washing it out from culture media. We therefore conclude that NB-DGJ might be a promising therapeutic chemical chaperone in infantile GM1 amenable variants and therefore warrants further analysis for its clinical applications.


Assuntos
1-Desoxinojirimicina/farmacologia , Fibroblastos/metabolismo , Gangliosidose GM1/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , beta-Galactosidase/metabolismo , 1-Desoxinojirimicina/química , Pré-Escolar , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Gangliosidose GM1/tratamento farmacológico , Gangliosidose GM1/patologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Chaperonas Moleculares/farmacologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Conformação Proteica , Transporte Proteico , beta-Galactosidase/química , beta-Galactosidase/genética
14.
PLoS One ; 15(3): e0230052, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214327

RESUMO

Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.


Assuntos
Doenças Autoimunes/patologia , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Osteocondrodisplasias/patologia , Fosfatase Ácida Resistente a Tartarato/metabolismo , Substituição de Aminoácidos , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Glicosilação , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/genética , Proteólise , Fosfatase Ácida Resistente a Tartarato/química , Fosfatase Ácida Resistente a Tartarato/genética
15.
Nat Chem Biol ; 16(6): 653-659, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32152544

RESUMO

Defining the biologically active structures of proteins in their cellular environments remains challenging for proteins with multiple conformations and functions, where only a minor conformer might be associated with a given function. Here, we use deep mutational scanning to probe the structure and dynamics of α-synuclein, a protein known to adopt disordered, helical and amyloid conformations. We examined the effects of 2,600 single-residue substitutions on the ability of intracellularly expressed α-synuclein to slow the growth of yeast. Computational analysis of the data showed that the conformation responsible for this phenotype is a long, uninterrupted, amphiphilic helix with increasing dynamics toward the C terminus. Deep mutational scanning can therefore determine biologically active conformations in cellular environments, even for a highly dynamic multi-conformational protein.


Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , alfa-Sinucleína/química , alfa-Sinucleína/genética , Sequência de Aminoácidos , Amiloide/química , Biblioteca Genômica , Modelos Moleculares , Fenótipo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Leveduras/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(15): 8563-8572, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32220963

RESUMO

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3 xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a ß-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3 xm.


Assuntos
Linfócitos B/imunologia , Linfopoese , Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Linfócitos T/imunologia , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Cristalografia por Raios X , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/imunologia , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Conformação Proteica , Linfócitos T/metabolismo , Linfócitos T/patologia
17.
PLoS One ; 15(2): e0229254, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074129

RESUMO

Activins are members of the transforming growth factor-ß (TGF-ß) superfamily of signaling proteins and were originally identified as components of follicular fluid. The proteins are now known to play critical roles in numerous normal and pathological processes and conditions, but less is clear about the relationships between their gene organization and protein variant expression and structure. The four human and mouse activin (Act) genes, termed INHßA, INHßB, INHßC and INHßE, differ in exon numbers. Human INHßA is the most complex with 7 exons and elicits production of three Act A variants (Act A X1, X2 and X3) differing in their pro-region, as we showed previously. Here we further analyzed the mouse INHßA gene and found that its 4 exons encode for a single open reading frame (mouse Act A), corresponding to the shortest human Act A X3 variant. Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature C- terminal ligand and are known to interact with the heparan sulfate (HS) chains of cell surface and matrix proteoglycans. Human Act A X1 and X2 variants do have a HS-binding domain (HBD) with Cardin/Weintraub traits in their pro-region, while the X3 variant does not as shown previously. We found that the mouse Act A lacks a HBD as well. However, we identified a typical HBD in the pro-region of both mouse and human Act B, and synthetic peptides containing that domain interacted with immobilized HS and cell surface with nanomolar affinity. In sum, human and mouse Act A genes elicit expression of different variant sets, while there is concordance in Act B protein expression, reflecting possible evolutionary diversity in function of, and responses to, these signaling proteins in the two species.


Assuntos
Ativinas/metabolismo , Variação Genética , Heparitina Sulfato/metabolismo , Proteínas Mutantes/metabolismo , Ativinas/química , Ativinas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Ligação Proteica , Conformação Proteica , Homologia de Sequência
18.
Biochem J ; 477(4): 787-800, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32011657

RESUMO

Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the cofactor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Arginina/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Ligação Proteica , Conformação Proteica , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética
19.
Biochem Biophys Res Commun ; 524(1): 123-128, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980172

RESUMO

Circadian rhythms are the endogenous oscillation of biological reactions and behaviors in most organisms on Earth. Circadian clocks are the pacemakers regulating circadian rhythms, and the transcription-translation dependent feedback loop (TTFL) model was supposed to be the sole model of circadian clocks. However, recent years have witnessed rapid progresses in the study of non-TTFL circadian clocks. The cyanobacterial circadian clock consists of three proteins (KaiA, KaiB, and KaiC), and is extensively studied as a non-TTFL circadian clock model. Although containing only three proteins, the molecular mechanism of the KaiABC circadian clock remains elusive. We recently noticed that KaiA has an auto-inhibition conformation during the oscillation, but how this auto-inhibition is regulated is unclear. Here, we started from the design of light modulated KaiAs to investigate this mechanism. We designed different KaiA constructs fused with the light modulable LOV2 protein, and used light-modulated KaiAs to regulate the phosphorylation and dephosphorylation of KaiC. Our data indicated that the N-terminal domain of KaiA is important for KaiA's reversible off/on switching during the unidirectional oscillation of the KaiABC system. This work provides an updated model to explain the molecular mechanism of the KaiABC circadian clock.


Assuntos
Proteínas de Bactérias/metabolismo , Relógios Circadianos/fisiologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Cianobactérias/fisiologia , Ativadores de Enzimas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Ritmo Circadiano/fisiologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , RNA Bacteriano
20.
Biochem Biophys Res Commun ; 524(1): 103-108, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980174

RESUMO

SP_0782 from Streptococcus pneumoniae is a dimeric PC4-like protein binding single-stranded DNA (ssDNA), and is potentially involved in maintenance of genome stability and natural transformation. SP_0782 binds with different lengths of ssDNA in various patterns through accommodating nucleotides differently in its two DNA-binding regions (DBRs). Here, we report the characterization of a novel site, leucine 20 (L20), which is not located in the DBRs but impairs the DNA binding when mutated to alanine (L20A). The L20A mutation markedly reduced the DNA-binding affinity of SP_0782 for ssDNA dT19G1, and affected the formation of high-order SP_0782:dT19G1 complexes. The side chain of L20 shows interactions with several residues at the backside of the DBRs in apo SP_0782 structure, and the L20A mutation led to a change of circular dichroism (CD) spectrum and broad chemical shift perturbations (CSPs) in NMR spectrum compared with the wild type. The most affected residues in NMR spectrum included F39 and R49 located in DBR2, as well as K60 in DBR1, which was suggested to be important for cooperative binding of ssDNA by the two subunits in SP_0782 dimer. Thus, the L20A mutation caused a local conformational change of SP_0782, which exerted an indirect effect on the DNA-binding interface and therefore impaired the affinity for ssDNA dT19G1. Interestingly, this L20 site is conserved in bacterial but not eukaryotic PC4-like proteins, suggesting an evolutionary divergence. This study provides an insight into the structure-function relationship of SP_0782, and an amino-acid site probably targeted for inhibiting bacteria selectively.


Assuntos
Proteínas de Bactérias/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Leucina/química , Proteínas Mutantes/química , Streptococcus pneumoniae/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Conformação Molecular , Proteínas Mutantes/genética , Mutação , Ligação Proteica
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