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1.
Biosensors (Basel) ; 11(5)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065240

RESUMO

Exosomes are a kind of membrane-bound phospholipid nanovesicle that are secreted extensively in a variety of biological fluids. Accumulating evidence has indicated that exosomes not only communicate with cells, but also perform functional roles in physiology and pathology. In addition, exosomes have also elicited a great deal of excitement due to their potential as disease biomarkers. Therefore, requirements for sensitive methods capable of precisely and specifically determining exosomes were needed. Herein, we not only develop a sensing surface to capture exosomes but also compare two surface proteins on exosomes, which are appropriate for detecting exosome surface markers by total internal reflected imaging ellipsometry (TIRIE). Protein G and antibody were immobilized on a thin layer of golden substrate to form the biosensing surface. The bio-interaction between antibodies and exosomes was recorded by the TIRIE in real time. The distance between exosomes adhered on a surface was 44 nm ± 0.5 nm. The KD  of anti-CD9 and exosome was lower than anti-CD63 and exosome by introducing pseudo-first-order interaction kinetics, which suggested that CD9 is more suitable for exosome surface markers than CD63. The limit of detection (LOD) of TIRIE was 0.4 µg/mL. In conclusion, we have proposed a surface for the detection of exosomes based on TIRIE, which can make the detection of exosomes convenient and efficient.


Assuntos
Biomarcadores , Técnicas Biossensoriais , Exossomos/química , Linhagem Celular Tumoral , Espectroscopia Dielétrica , Humanos , Limite de Detecção , Proteínas de Membrana , Ligação Proteica
2.
MAbs ; 13(1): 1919285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34074219

RESUMO

The newly emerging variants of SARS-CoV-2 from South Africa (B.1.351/501Y.V2) and Brazil (P.1/501Y.V3) have led to a higher infection rate and reinfection of COVID-19 patients. We found that the mutations K417N, E484K, and N501Y within the receptor-binding domains (RBDs) of the virus could confer ~2-fold higher binding affinity to the human receptor, angiotensin converting enzyme 2 (ACE2), compared to the wildtype RBD. The mutated version of RBD also completely abolishes the binding of bamlanivimab, a therapeutic antibody, in vitro. Detailed analysis shows that the ~10-fold gain of binding affinity between ACE2 and Y501-RBD, which also exits in the high contagious variant B.1.1.7/501Y.V1 from the United Kingdom, is compromised by additional introduction of the K417/N/T mutation. Mutation of E484K leads to the loss of bamlanivimab binding to RBD, although this mutation does not affect the binding between RBD and ACE2.


Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Antivirais/metabolismo , COVID-19/virologia , Mutação , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/diagnóstico , COVID-19/tratamento farmacológico , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética
3.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064454

RESUMO

The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR-D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.


Assuntos
Potenciação de Longa Duração/genética , Neurônios/metabolismo , Receptor Cross-Talk , Receptores de Dopamina D5/genética , Receptores de GABA-A/genética , Transmissão Sináptica/genética , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Peptídeos/síntese química , Peptídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D5/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/genética , Sinapses/metabolismo
4.
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
5.
Nat Commun ; 12(1): 3396, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099711

RESUMO

Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with overlapping clinical features and the pathological hallmark of cytoplasmic deposits of misfolded proteins. The most frequent cause of familial forms of these diseases is a hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene that is translated into dipeptide repeat polymers. Here we show that proline/arginine repeat polymers derail protein folding by sequestering molecular chaperones. We demonstrate that proline/arginine repeat polymers inhibit the folding catalyst activity of PPIA, an abundant molecular chaperone and prolyl isomerase in the brain that is altered in amyotrophic lateral sclerosis. NMR spectroscopy reveals that proline/arginine repeat polymers bind to the active site of PPIA. X-ray crystallography determines the atomic structure of a proline/arginine repeat polymer in complex with the prolyl isomerase and defines the molecular basis for the specificity of disease-associated proline/arginine polymer interactions. The combined data establish a toxic mechanism that is specific for proline/arginine dipeptide repeat polymers and leads to derailed protein homeostasis in C9orf72-associated neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Proteína C9orf72/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/patologia , Peptidilprolil Isomerase/metabolismo , Esclerose Amiotrófica Lateral/genética , Arginina/genética , Arginina/metabolismo , Biopolímeros/metabolismo , Encéfalo/patologia , Domínio Catalítico , Cristalografia por Raios X , Expansão das Repetições de DNA , Dipeptídeos/genética , Demência Frontotemporal/genética , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptidilprolil Isomerase/isolamento & purificação , Peptidilprolil Isomerase/ultraestrutura , Prolina/genética , Prolina/metabolismo , Agregados Proteicos/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Sequências Repetitivas de Aminoácidos/genética
6.
Nat Commun ; 12(1): 3451, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103486

RESUMO

Several cell-surface receptors for neurotoxic forms of amyloid-ß (Aß) have been described, but their molecular interactions with Aß assemblies and their relative contributions to mediating Alzheimer's disease pathology have remained uncertain. Here, we used super-resolution microscopy to directly visualize Aß-receptor interactions at the nanometer scale. We report that one documented Aß receptor, PrPC, specifically inhibits the polymerization of Aß fibrils by binding to the rapidly growing end of each fibril, thereby blocking polarized elongation at that end. PrPC binds neurotoxic oligomers and protofibrils in a similar fashion, suggesting that it may recognize a common, end-specific, structural motif on all of these assemblies. Finally, two other Aß receptors, FcγRIIb and LilrB2, affect Aß fibril growth in a manner similar to PrPC. Our results suggest that receptors may trap Aß oligomers and protofibrils on the neuronal surface by binding to a common molecular determinant on these assemblies, thereby initiating a neurotoxic signal.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Neurotoxinas/química , Multimerização Proteica , Receptores de Superfície Celular/metabolismo , Animais , Benzotiazóis/metabolismo , Calmodulina/metabolismo , Humanos , Cinética , Camundongos , Modelos Biológicos , Polimerização , Príons/metabolismo , Ligação Proteica , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo
7.
Nat Commun ; 12(1): 3456, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103492

RESUMO

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.


Assuntos
Proteínas de Transporte/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Dedos de Zinco , Sítios de Ligação , Proteínas de Transporte/química , Sobrevivência Celular , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Schizosaccharomyces/citologia
8.
Nat Commun ; 12(1): 3469, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103513

RESUMO

In most bacteria, cell division depends on the tubulin homolog FtsZ and other proteins, such as SepF, that form a complex termed the divisome. Cell division also depends on FtsZ in many archaea, but other components of the divisome are unknown. Here, we demonstrate that a SepF homolog plays important roles in cell division in Haloferax volcanii, a halophilic archaeon that is known to have two FtsZ homologs with slightly different functions (FtsZ1 and FtsZ2). SepF co-localizes with both FtsZ1 and FtsZ2 at midcell. Attempts to generate a sepF deletion mutant were unsuccessful, suggesting an essential role. Indeed, SepF depletion leads to severe cell division defects and formation of large cells. Overexpression of FtsZ1-GFP or FtsZ2-GFP in SepF-depleted cells results in formation of filamentous cells with a high number of FtsZ1 rings, while the number of FtsZ2 rings is not affected. Pull-down assays support that SepF interacts with FtsZ2 but not with FtsZ1, although SepF appears delocalized in the absence of FtsZ1. Archaeal SepF homologs lack a glycine residue known to be important for polymerization and function in bacteria, and purified H. volcanii SepF forms dimers, suggesting that polymerization might not be important for the function of archaeal SepF.


Assuntos
Proteínas Arqueais/metabolismo , Divisão Celular , Haloferax volcanii/citologia , Haloferax volcanii/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Forma Celular , Proteínas de Fluorescência Verde/metabolismo , Haloferax volcanii/crescimento & desenvolvimento , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Triptofano/deficiência
9.
Nat Commun ; 12(1): 3426, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103516

RESUMO

Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Complexos Multiproteicos/metabolismo , Transcrição Genética , Proteínas de Arabidopsis/genética , Epistasia Genética , Genes de Plantas , Loci Gênicos , Fatores de Transcrição de Choque Térmico/genética , Histonas/metabolismo , Cinética , Lisina/metabolismo , Metilação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
10.
Nat Commun ; 12(1): 3428, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103526

RESUMO

Dysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.


Assuntos
Aborto Habitual/metabolismo , Vilosidades Coriônicas/metabolismo , Ácido Succínico/metabolismo , Aborto Habitual/enzimologia , Aborto Habitual/genética , Animais , Estudos de Casos e Controles , Hipóxia Celular , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaboloma , Camundongos Endogâmicos C57BL , Gravidez , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Risco , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Trofoblastos/metabolismo , Trofoblastos/patologia
11.
Nat Commun ; 12(1): 3444, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103528

RESUMO

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.


Assuntos
Arginina/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Metilação/efeitos dos fármacos , Camundongos Nus , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína-Arginina N-Metiltransferases/deficiência , Proteínas Proto-Oncogênicas c-akt/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066122

RESUMO

The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q's CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.


Assuntos
Colágeno/metabolismo , Complemento C1q/metabolismo , Receptores Imunológicos/metabolismo , Sítios de Ligação , Complemento C1q/genética , Humanos , Tolerância Imunológica , Mutação , Ligação Proteica , Receptores Imunológicos/genética
13.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067978

RESUMO

Galectin-3 (Gal-3) is a ß-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a ß-galactoside LacdiNAc (GalNAcß1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by ß1 and αV integrins-namely α5ß1, αVß3, and αVß1 integrins.


Assuntos
Proteínas Sanguíneas/metabolismo , Adesão Celular , Junções Célula-Matriz/metabolismo , Galectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Integrinas/metabolismo , Células-Tronco Mesenquimais/fisiologia , Sítios de Ligação , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Ligação Proteica
14.
Bioengineered ; 12(1): 2274-2287, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34077310

RESUMO

Xuebijing Injection have been found to improve the clinical symptoms of COVID-19 and alleviate disease severity, but the mechanisms are currently unclear. This study aimed to investigate the potential molecular targets and mechanisms of the Xuebijing injection in treating COVID-19 via network pharmacology and molecular docking analysis. The main active ingredients and therapeutic targets of the Xuebijing injection, and the pathogenic targets of COVID-19 were screened using the TCMSP, UniProt, and GeneCard databases. According to the 'Drug-Ingredients-Targets-Disease' network built by STRING and Cytoscape, AKT1 was identified as the core target, and baicalein, luteolin, and quercetin were identified as the active ingredients of the Xuebijing injection in connection with AKT1. R language was used for enrichment analysis that predict the mechanisms by which the Xuebijing injection may inhibit lipopolysaccharide-mediated inflammatory response, modulate NOS activity, and regulate the TNF signal pathway by affecting the role of AKT1. Based on the results of network pharmacology, a molecular docking was performed with AKT1 and the three active ingredients, the results indicated that all three active ingredients could stably bind with AKT1. These findings identify potential molecular mechanisms by which Xuebijing Injection inhibit COVID-19 by acting on AKT1.


Assuntos
Antivirais/administração & dosagem , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , SARS-CoV-2 , Antivirais/farmacocinética , Antivirais/farmacologia , Engenharia Biomédica , Medicamentos de Ervas Chinesas/farmacocinética , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/administração & dosagem , Humanos , Injeções , Luteolina/administração & dosagem , Simulação de Acoplamento Molecular , Pandemias , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/administração & dosagem , Transdução de Sinais/efeitos dos fármacos
15.
J Phys Chem B ; 125(21): 5455-5457, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34078077
16.
Nat Commun ; 12(1): 3214, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088904

RESUMO

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZCTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.


Assuntos
Proteínas Arqueais/metabolismo , Divisão Celular/fisiologia , Methanobrevibacter/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular , Divisão Celular/genética , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Methanobrevibacter/genética , Methanobrevibacter/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
17.
Viruses ; 13(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067878

RESUMO

COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.


Assuntos
Enzima de Conversão de Angiotensina 2/química , COVID-19/virologia , Polissacarídeos/química , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Sítios de Ligação , Glicosilação , Interações entre Hospedeiro e Microrganismos , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ligação Viral
18.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070162

RESUMO

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other's activity, indicating an essential role of protein-RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein-RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein-RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein-RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein-RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.


Assuntos
Imunoprecipitação/métodos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação/tendências , Ligação Proteica , RNA Polimerase II/metabolismo , Transcrição Genética , Transcriptoma
19.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
20.
Nat Commun ; 12(1): 3292, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078910

RESUMO

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.


Assuntos
Autofagia/genética , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Cadeias Pesadas de Miosina/genética , Quinases Relacionadas a NIMA/genética , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Cílios/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Quinases Relacionadas a NIMA/deficiência , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
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