Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 59.128
Filtrar
1.
Nat Commun ; 12(1): 4721, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354057

RESUMO

G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT1 receptor) - a prototypical class A GPCR-activation. Our findings suggest a synergistic transition mechanism for AT1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Mutation of this cryptic site prevents activation of the downstream G protein signaling and ß-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. Together, these findings provide a deeper understanding of AT1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.


Assuntos
Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação/genética , Desenho de Fármacos , Humanos , Cadeias de Markov , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais , beta-Arrestinas/metabolismo
2.
Nat Commun ; 12(1): 4709, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354080

RESUMO

Allostery represents a fundamental mechanism of biological regulation that involves long-range communication between distant protein sites. It also provides a powerful framework for novel therapeutics. NMDA receptors (NMDARs), glutamate-gated ionotropic receptors that play central roles in synapse maturation and plasticity, are prototypical allosteric machines harboring large extracellular N-terminal domains (NTDs) that provide allosteric control of key receptor properties with impact on cognition and behavior. It is commonly thought that GluN2A and GluN2B receptors, the two predominant NMDAR subtypes in the adult brain, share similar allosteric transitions. Here, combining functional and structural interrogation, we reveal that GluN2A and GluN2B receptors utilize different long-distance allosteric mechanisms involving distinct subunit-subunit interfaces and molecular rearrangements. NMDARs have thus evolved multiple levels of subunit-specific allosteric control over their transmembrane ion channel pore. Our results uncover an unsuspected diversity in NMDAR molecular mechanisms with important implications for receptor physiology and precision drug development.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fotoquímica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
3.
Nat Commun ; 12(1): 4476, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294706

RESUMO

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.


Assuntos
Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Edição de Genes , Bacteriófagos/enzimologia , Bacteriófagos/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Clivagem do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , RNA Guia/genética , RNA Guia/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
4.
Nat Commun ; 12(1): 4452, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294713

RESUMO

Atherosclerosis-associated cardiovascular disease is one of the main causes of death and disability among patients with diabetes mellitus. However, little is known about the impact of S-nitrosylation in diabetes-accelerated atherosclerosis. Here, we show increased levels of S-nitrosylation of guanine nucleotide-binding protein G(i) subunit alpha-2 (SNO-GNAI2) at Cysteine 66 in coronary artery samples from diabetic patients with atherosclerosis, consistently with results from mice. Mechanistically, SNO-GNAI2 acted by coupling with CXCR5 to dephosphorylate the Hippo pathway kinase LATS1, thereby leading to nuclear translocation of YAP and promoting an inflammatory response in endothelial cells. Furthermore, Cys-mutant GNAI2 refractory to S-nitrosylation abrogated GNAI2-CXCR5 coupling, alleviated atherosclerosis in diabetic mice, restored Hippo activity, and reduced endothelial inflammation. In addition, we showed that melatonin treatment restored endothelial function and protected against diabetes-accelerated atherosclerosis by preventing GNAI2 S-nitrosylation. In conclusion, SNO-GNAI2 drives diabetes-accelerated atherosclerosis by coupling with CXCR5 and activating YAP-dependent endothelial inflammation, and reducing SNO-GNAI2 is an efficient strategy for alleviating diabetes-accelerated atherosclerosis.


Assuntos
Aterosclerose/etiologia , Aterosclerose/metabolismo , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Cisteína/química , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/química , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Melatonina/farmacologia , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Óxido Nítrico Sintase Tipo II/metabolismo , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores CXCR5/deficiência , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Fatores de Transcrição/metabolismo
5.
J Chem Theory Comput ; 17(8): 5301-5311, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34270241

RESUMO

Though crucial for understanding the function of large biomolecular systems, locating the minimum free energy paths (MFEPs) between their key conformational states is far from trivial due to their high-dimensional nature. Most existing path-searching methods require a static collective variable space as input, encoding intuition or prior knowledge of the transition mechanism. Such information is, however, hardly available a priori and expensive to validate. To alleviate this issue, we have previously introduced a Traveling-salesman based Automated Path Searching method (TAPS) and demonstrated its efficiency on simple peptide systems. Having implemented a parallel version of this method, here we assess the performance of TAPS on three realistic systems (tens to hundreds of residues) in explicit solvents. We show that TAPS successfully located the MFEP for the ground/excited state transition of the T4 lysozyme L99A variant, consistent with previous findings. TAPS also helped identifying the important role of the two polar contacts in directing the loop-in/loop-out transition of the mitogen-activated protein kinase kinase (MEK1), which explained previous mutant experiments. Remarkably, at a minimal cost of 126 ns sampling, TAPS revealed that the Ltn40/Ltn10 transition of lymphotactin needs no complete unfolding/refolding of its ß-sheets and that five polar contacts are sufficient to stabilize the various partially unfolded intermediates along the MFEP. These results present TAPS as a general and promising tool for studying the functional dynamics of complex biomolecular systems.


Assuntos
MAP Quinase Quinase 1/química , Muramidase/química , Linfocinas/química , Linfocinas/metabolismo , MAP Quinase Quinase 1/metabolismo , Simulação de Dinâmica Molecular , Muramidase/genética , Muramidase/metabolismo , Mutagênese Sítio-Dirigida , Conformação Proteica em Folha beta , Desdobramento de Proteína , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismo
6.
Methods Mol Biol ; 2287: 199-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270031

RESUMO

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.


Assuntos
Endonucleases/metabolismo , Haploidia , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Mutagênese Sítio-Dirigida/métodos , Melhoramento Vegetal/métodos , RNA Guia/genética , Sistemas CRISPR-Cas , Meios de Cultura , Endonucleases/genética , Edição de Genes , Engenharia Genética , Genoma de Planta , Homozigoto , Hordeum/embriologia , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento
7.
Am J Hum Genet ; 108(9): 1735-1751, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34314704

RESUMO

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Mutação de Sentido Incorreto , Medicamentos sob Prescrição/metabolismo , Saccharomyces cerevisiae/enzimologia , Xenobióticos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/genética , Ensaios Enzimáticos , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenitoína/química , Polimorfismo Genético , Medicamentos sob Prescrição/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Transgenes , Varfarina/química , Varfarina/metabolismo , Xenobióticos/química
8.
Cell Mol Life Sci ; 78(17-18): 6305-6318, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34292354

RESUMO

The structural basis for the GTPase-accelerating activity of regulators of G protein signaling (RGS) proteins, as well as the mechanistic basis for their specificity in interacting with the heterotrimeric (αßγ) G proteins they inactivate, is not sufficiently understood at the family level. Here, we used biochemical assays to compare RGS domains across the RGS family and map those individual residues that favorably contribute to GTPase-accelerating activity, and those residues responsible for attenuating RGS domain interactions with Gα subunits. We show that conserved interactions of RGS residues with both the Gα switch I and II regions are crucial for RGS activity, while the reciprocal effects of "modulatory" and "disruptor" residues selectively modulate RGS activity. Our results quantify how specific interactions between RGS domains and Gα subunits are set by a balance between favorable RGS residue interactions with particular Gα switch regions, and unfavorable interactions with the Gα helical domain.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Alinhamento de Sequência , Termodinâmica
9.
Int J Biol Macromol ; 185: 949-958, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34237366

RESUMO

Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii, we observed that Ses-TwTPS1-1 and Ses-TwTPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses-TwTPS1-2, Ses-TwTPS3, and Ses-TwTPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E, E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses-TwTPS1-1:V199I and Ses-TwTPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Tripterygium/enzimologia , Alquil e Aril Transferases/química , Domínio Catalítico , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Terpenos/metabolismo , Tripterygium/genética
10.
Org Biomol Chem ; 19(30): 6650-6656, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34264250

RESUMO

The exquisite chemodiversity of terpenoids is the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue (Cys440 in Nicotiana tabacum 5-epi-aristolochene synthase) proximal to an ion-binding motif common to all TPSs and named the preNSE/DTE residue, which determines the product specificity of sesquiterpene synthases from different plant species. In sesquiterpene synthases catalyzing 1,10-cyclization (1,10-cyclases) of farnesyl diphosphate, mutation of the residue in both specific and promiscuous 1,10-cyclases from different lineages leads to the accumulation of monocyclic germacrene A-11-ol, which is "short-circuited" from complex cyclization cascades, suggesting a key role of this residue in generating the first common intermediate of 1,10-cyclization. Altering this residue in a specific 1,11-cyclase results in alternative 1,10-cyclization products. Moreover, the preNSE/DTE residue can be harnessed to engineer highly specific sesquiterpene synthases for an improved proportion of high-value terpenoids, such as patchoulol, a main constituent of several traditional Chinese medicines that could treat SARS-CoV-2.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Biocatálise , Alquil e Aril Transferases/genética , Domínio Catalítico , Ciclização , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Tabaco/enzimologia
11.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299011

RESUMO

Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.


Assuntos
Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Fraturas do Fêmur/genética , Fraturas do Fêmur/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Citocromo P-450 CYP1A1/química , Difosfonatos/uso terapêutico , Fraturas do Fêmur/enzimologia , Humanos , Incidência , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Filogenia , Alinhamento de Sequência
12.
Nat Commun ; 12(1): 4417, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285212

RESUMO

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.


Assuntos
Aspergillus/enzimologia , Biocatálise , Proteínas Fúngicas/metabolismo , Oxigenases/metabolismo , Peróxidos/metabolismo , Aspergillus/genética , Domínio Catalítico , Cristalografia por Raios X , Compostos Ferrosos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Ácidos Cetoglutáricos/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/metabolismo , Oxigenases/genética , Oxigenases/isolamento & purificação , Oxigenases/ultraestrutura , Conformação Proteica em Folha beta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Terpenos/metabolismo
13.
FASEB J ; 35(8): e21681, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196428

RESUMO

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.


Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
14.
Nat Commun ; 12(1): 4223, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244518

RESUMO

The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor and template for flagellar assembly. The C ring attached to the MS ring is involved in torque generation and rotation switch, and a large symmetry mismatch between these two rings has been a long puzzle, especially with respect to their role in motor function. Here, using cryoEM structural analysis of the flagellar basal body and the MS ring formed by full-length FliF from Salmonella enterica, we show that the native MS ring is formed by 34 FliF subunits with no symmetry variation. Symmetry analysis of the C ring shows a variation with a peak at 34-fold, suggesting flexibility in C ring assembly. Finally, our data also indicate that FliF subunits assume two different conformations, contributing differentially to the inner and middle parts of the M ring and thus resulting in 23- and 11-fold subsymmetries in the inner and middle M ring, respectively. The internal core of the M ring, formed by 23 subunits, forms a hole of the right size to accommodate the protein export gate.


Assuntos
Proteínas de Bactérias/ultraestrutura , Flagelos/ultraestrutura , Proteínas de Membrana/ultraestrutura , Sistemas de Secreção Tipo III/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Fracionamento Celular , Microscopia Crioeletrônica , Flagelos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/ultraestrutura , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
15.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210100

RESUMO

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Edição de Genes/métodos , Proteínas de Plantas/metabolismo , Triticale/metabolismo , Sistemas CRISPR-Cas , Sistema Enzimático do Citocromo P-450/genética , Genes Reporter , Mutação INDEL , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Transfecção , Triticale/genética
16.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200573

RESUMO

In recent years, cyclic guanosine 3',5'-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules-BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP.


Assuntos
Brachypodium/enzimologia , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Mutação , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Guanilato Ciclase/química , Guanilato Ciclase/genética , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Conformação Proteica , Homologia de Sequência , Transdução de Sinais
17.
Nat Commun ; 12(1): 3266, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075032

RESUMO

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.


Assuntos
Infecções por Flavivirus/terapia , Flavivirus/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Vírion/ultraestrutura , Aedes/virologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica , Dengue/terapia , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/farmacologia , Desenho de Fármacos , Flavivirus/efeitos dos fármacos , Flavivirus/imunologia , Flavivirus/patogenicidade , Infecções por Flavivirus/virologia , Humanos , Mesocricetus , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Células Vero , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/farmacologia , Vacinas Virais/uso terapêutico , Vírion/efeitos dos fármacos , Vírion/metabolismo
18.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069970

RESUMO

Prostate cancer (PCa) is the second most leading cause of death in males. Our previous studies have demonstrated that δ-catenin plays an important role in prostate cancer progression. However, the molecular mechanism underlying the regulation of δ-catenin has not been fully explored yet. In the present study, we found that δ-catenin could induce phosphorylation of p21Waf and stabilize p21 in the cytoplasm, thus blocking its nuclear accumulation for the first time. We also found that δ-catenin could regulate the interaction between AKT and p21, leading to phosphorylation of p21 at Thr-145 residue. Finally, EGF was found to be a key factor upstream of AKT/δ-catenin/p21 for promoting proliferation and metastasis in prostate cancer. Our findings provide new insights into molecular controls of EGF and the development of potential therapeutics targeting δ-catenin to control prostate cancer progression.


Assuntos
Cateninas/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/química , Inibidor de Quinase Dependente de Ciclina p21/genética , Humanos , Ligantes , Masculino , Modelos Biológicos , Mutagênese Sítio-Dirigida , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Células PC-3 , Fosforilação , Neoplasias da Próstata/genética , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/química , Transdução de Sinais , Treonina/química
19.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070125

RESUMO

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of ß-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused ß-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in ß-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced ß-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by ß-arrestins and that a highly conserved tyrosine residue within MRGPRX2's NPxxY motif contributes to both G protein- and ß-arrestin-mediated responses.


Assuntos
Proteínas do Tecido Nervoso/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropeptídeos/agonistas , Substância P/metabolismo , beta-Arrestinas/metabolismo , Substituição de Aminoácidos , Animais , Degranulação Celular , Linhagem Celular , Feminino , Humanos , Masculino , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neuroimunomodulação/fisiologia , Ratos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Tirosina/química , beta-Arrestina 2/deficiência , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
20.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...