Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.704
Filtrar
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): 3690, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704140

RESUMO

Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. Our structures, in combination with functional analyses, delineate a structural mechanism by which mechanosensitive channels open under increased membrane tension. Further, the shared structural feature between unrelated channels suggests the possibility of a unified mechanical gating mechanism stemming from membrane deformation induced by a non-planar transmembrane domain.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Eucariotos/metabolismo , Ativação do Canal Iônico , Mecanotransdução Celular , Proteínas de Arabidopsis/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
3.
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
4.
Science ; 369(6506): 1014-1018, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32540904

RESUMO

Antibodies targeting the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present a promising approach to combat the coronavirus disease 2019 (COVID-19) pandemic; however, concerns remain that mutations can yield antibody resistance. We investigated the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared after in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Escape mutants were not generated after treatment with a noncompeting antibody cocktail.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Betacoronavirus/química , Betacoronavirus/genética , Epitopos , Genoma Viral , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/imunologia , Mutação , Testes de Neutralização , Pandemias , Domínios e Motivos de Interação entre Proteínas , Seleção Genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
5.
Nat Commun ; 11(1): 3228, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591529

RESUMO

Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.


Assuntos
5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Biocatálise , Plasmodium falciparum/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/metabolismo , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas Mutantes/química , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Especificidade por Substrato
6.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140464, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32497661

RESUMO

The residual solution structures of two alpha-synuclein mutants, A30P and A53T, observed in family members of patients with Parkinson's disease were compared with that of wild-type by NMR. The A53T substitution had been shown to accelerate fibril formation of alpha-synuclein, whereas the A30P mutation has the negative and positive effects on the formation of the fibril and spherical oligomer, respectively. The remaining structure was analyzed via amide-proton exchange and signal intensity measurements using NMR. Amide-proton exchange was used for both the calculation of kex values and ratio of kex at different temperatures. Effects of the A30P (N-terminal region) mutation were observed at the C-terminal region as a more flexible structure, suggesting that long-range interactions exist between the N- and C-terminal regions in alpha-synuclein. In addition, the N-terminal region adopted a more rigid structure in the A53T and A30P mutants than in the wild-type. It was concluded that the structural change caused by the mutations is related to the formation of a beta-hairpin at the initiation site of the N-terminal core structure. Furthermore, the signal intensity was used to estimate the rigidity of the structure. Higher signal intensities were observed for A30P at the 112, 113, and 116 C-terminal residues, suggesting that this region adopts more flexible structure. The ratio of the intensities at different temperatures indicated more flexible or rigid structures in the N-terminal region of A30P than in that of wild-type. Thus, using different approaches and temperatures is a good method to analyze residual structure in intrinsically disordered proteins.


Assuntos
Amidas/química , Proteínas Intrinsicamente Desordenadas/genética , Prótons , alfa-Sinucleína/química , Humanos , Proteínas Intrinsicamente Desordenadas/química , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/química , Mutação , Doença de Parkinson/genética , Estrutura Secundária de Proteína , Temperatura , alfa-Sinucleína/genética
7.
Nat Commun ; 11(1): 2820, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499486

RESUMO

As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn's aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.


Assuntos
Agregados Proteicos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Benzotiazóis/metabolismo , Cálcio/metabolismo , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Fosforilação , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-Atividade
8.
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
9.
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
10.
Science ; 369(6505): 806-811, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32434945

RESUMO

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/fisiologia , Líquido da Lavagem Broncoalveolar/virologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Secundária , Imunogenicidade da Vacina , Memória Imunológica , Macaca mulatta , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/imunologia , Mucosa Nasal/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Vacinas de DNA/administração & dosagem , Carga Viral , Vacinas Virais/administração & dosagem
11.
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
12.
PLoS One ; 15(4): e0231177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271820

RESUMO

Enrichment of omega-3 fatty acids (É·-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky É·-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the É·-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity.


Assuntos
Ácidos Graxos Ômega-3/metabolismo , Geobacillus/enzimologia , Lipase/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Aminoácidos/metabolismo , Domínio Catalítico , Simulação por Computador , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/química , Lipase/isolamento & purificação , Metilação , Modelos Moleculares , Proteínas Mutantes/análise , Proteínas Mutantes/química , Mutação/genética , Dobramento de Proteína , Especificidade por Substrato , Temperatura
13.
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
14.
Nat Commun ; 11(1): 1475, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193462

RESUMO

Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to these response differences, here we analyze in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis indicates that the p53 family protein, p73, affects Th1 differentiation. In cells differentiated under Th1 conditions in vitro, p73 negatively regulates IFNγ production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes such as Ifng and Il12rb2. Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice have increased IFNγ production and less disease severity, whereas in an adoptive transfer model of inflammatory bowel disease, transfer of p73-deficient naïve CD4+ T cells increases Th1 responses and augments disease severity. Our results thus identify p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease.


Assuntos
Diferenciação Celular , Células Th1/citologia , Células Th1/metabolismo , Proteína Tumoral p73/metabolismo , Alelos , Animais , Sequência de Bases , Sítios de Ligação , Colite/patologia , DNA/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Deleção de Genes , Regulação da Expressão Gênica , Interferon gama/metabolismo , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Índice de Gravidade de Doença , Proteína Tumoral p73/química , Proteína Tumoral p73/deficiência , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Biochim Biophys Acta Bioenerg ; 1861(7): 148190, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32194062

RESUMO

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm-1 and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm-1) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.


Assuntos
Luz , Retinaldeído/química , Rodopsina/química , Bases de Schiff/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Cristalografia por Raios X , Flavobacteriaceae/metabolismo , Ligação de Hidrogênio , Isomerismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Isótopos de Nitrogênio , Espectroscopia de Infravermelho com Transformada de Fourier , Vibração , Água/química
16.
J Chromatogr A ; 1620: 460986, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32173023

RESUMO

Human plasminogen Kringle 5 is known to pose a more potent anti-angiogenesis effect by inducing endothelial cell apoptosis. Our previous studies have identified the peptide IGNSNTL as a binding sequence of Kringle 5 using Ph.D.-7 phage display peptide library and enzyme-linked immunosorbent assay. Here, eleven proteins were screened and summarized by BLAST, laminin α3 chain G1 domain (LG1) was considered as the most potential receptor based on E value and domain function. The specific interaction of them was directly revealed through ligand blot and a strong concentration-dependent manner occurred between them (Ka 4.30 × 105 L mol-1) in frontal chromatography observation. Moreover, R10A/P83R substitution Kringle 5 decreased the affinity capacity to LG1. Furthermore, a remarkable conformational change from random coil3 to α helix and α1 helix to random coil were observed to the structural compactness and stability for LG1. Surface loops and coils also showed fluctuations up to some extent, giving the binding surface greater flexibility and correspondingly allowing for induced-fit binding, which was -23.87 kcal mol-1 of the free energy with electrostatic force as a main driver. Taken together, not only effective theoretical prediction and experiment validated that LG1 is receptor of Kringle 5, but also give an new perspective of the binding mechanism of Kringle 5 and its specific receptor and could facilitate the development of novel agent targeted toward pathologic angiogenesis.


Assuntos
Células Endoteliais/metabolismo , Laminina/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/metabolismo , Plasminogênio/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Cinética , Ligantes , Proteínas Mutantes/química , Biblioteca de Peptídeos , Ligação Proteica , Domínios Proteicos , Termodinâmica
17.
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
18.
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
19.
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
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA