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1.
Exp Parasitol ; 217: 107963, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32781092

RESUMO

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.


Assuntos
Eimeria tenella/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Ceco/parasitologia , Linhagem Celular , Embrião de Galinha , Galinhas , Biologia Computacional , DNA Complementar/genética , DNA Complementar/metabolismo , Eimeria tenella/química , Eletroforese em Gel de Poliacrilamida , Fezes/parasitologia , Fibroblastos , Técnica Indireta de Fluorescência para Anticorpo , Biossíntese de Proteínas , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Ribossômicas/química , Organismos Livres de Patógenos Específicos , Transcrição Genética
2.
Invest Ophthalmol Vis Sci ; 61(5): 13, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32396635

RESUMO

Purpose: Stimulated by evidence implicating diurnal/circadian rhythms and light in refractive development, we studied the expression over 24 hours of selected clock and circadian rhythm-related genes in retina/retinal pigment epithelium (RPE) and choroid of experimental ametropias in chicks. Methods: Newly hatched chicks, entrained to a 12-hour light/dark cycle for 12 to 14 days, either experienced nonrestricted vision OU (i.e., in both eyes) or received an image-blurring diffuser or a minus 10-diopter (D) or a plus 10-D defocusing lens over one eye. Starting 1 day later and at 4-hour intervals for 24 hours, the retina/RPE and choroid were separately dissected. Without pooling, total RNA was extracted, converted to cDNA, and assayed by quantitative PCR for the expression of the following genes: Opn4m, Clock, Npas2, Per3, Cry1, Arntl, and Mtnr1a. Results: The expression of each gene in retina/RPE and in choroid of eyes with nonrestricted vision OU varied over 24 hours, with equal levels OU for most genes and times. Altered visual input influenced gene expression in complex patterns that varied by gene, visual input, time, and eye, affecting experimental eyes with altered vision and also contralateral eyes with nonrestricted vision. Discussion: Altering visual input in ways known to induce ametropias alters the retinal/RPE and choroidal expression of circadian rhythm-related genes, further linking circadian biology with eye growth regulation. While further investigations are needed, studying circadian processes may help understand refractive mechanisms and the increasing myopia prevalence in contemporary societies where lighting patterns can desynchronize endogenous rhythms from the natural environmental light/dark cycle.


Assuntos
Corioide/metabolismo , Ritmo Circadiano/genética , Perfilação da Expressão Gênica , Erros de Refração/etiologia , Retina/metabolismo , Acuidade Visual , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Galinhas , Criptocromos/genética , Criptocromos/metabolismo , DNA Complementar/metabolismo , Escuridão , Modelos Animais de Doenças , Luz , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo
3.
Nat Commun ; 11(1): 1972, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332881

RESUMO

Shieldin, including SHLD1, SHLD2, SHLD3 and REV7, functions as a bridge linking 53BP1-RIF1 and single-strand DNA to suppress the DNA termini nucleolytic resection during non-homologous end joining (NHEJ). However, the mechanism of shieldin assembly remains unclear. Here we present the crystal structure of the SHLD3-REV7-SHLD2 ternary complex and reveal an unexpected C (closed)-REV7-O (open)-REV7 conformational dimer mediated by SHLD3. We show that SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 by forming ß sheet sandwich. Disruption of the REV7 conformational dimer abolishes the assembly of shieldin and impairs NHEJ efficiency. The conserved FXPWFP motif of SHLD3 binds to C-REV7 and blocks its binding to REV1, which excludes shieldin from the REV1/Pol ζ translesion synthesis (TLS) complex. Our study reveals the molecular architecture of shieldin assembly, elucidates the structural basis of the REV7 conformational dimer, and provides mechanistic insight into orchestration between TLS and NHEJ.


Assuntos
Proteínas de Ciclo Celular/química , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/química , Proteínas Mad2/química , Motivos de Aminoácidos , Cristalografia por Raios X , DNA Complementar/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína
4.
Nat Commun ; 11(1): 1774, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286321

RESUMO

Protein ubiquitination is one of the most prevalent post-translational modifications, controlling virtually every process in eukaryotic cells. Recently, the Legionella effector MavC was found to mediate a unique ubiquitination through transglutamination, linking ubiquitin (Ub) to UBE2N through UbGln40 in a process that can be inhibited by another Legionella effector, Lpg2149. Here, we report the structures of MavC/UBE2N/Ub ternary complex, MavC/UBE2N-Ub (product) binary complex, and MavC/Lpg2149 binary complex. During the ubiquitination, the loop containing the modification site K92 of UBE2N undergoes marked conformational change, and Lpg2149 inhibits this ubiquitination through competing with Ub to bind MavC. Moreover, we found that MavC itself also exhibits weak deubiquitinase activity towards this non-canonical ubiquitination. Together, our study not only provides insights into the mechanism and inhibition of this transglutaminase-induced ubiquitination by MavC, but also sheds light on the future studies into UBE2N inhibition by this modification and deubiquitinases of this unique ubiquitination.


Assuntos
Legionella/metabolismo , Transglutaminases/metabolismo , Ubiquitinação/fisiologia , Cristalografia por Raios X , DNA Complementar/metabolismo , Enzimas Desubiquitinantes/metabolismo , Humanos , Estrutura Secundária de Proteína , Ubiquitinação/genética
5.
Anim Reprod Sci ; 210: 106192, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31635778

RESUMO

This study was conducted to elucidate mare cervical dilation mechanisms by testing two hypotheses: (i) the proportion of collagen staining in histological samples of mare cervices and (ii) the abundance of hormone receptors in the equine cervix differ with stage of the oestrous cycle and site within the cervix. Tissues and jugular vein blood samples were collected from 15 mares. Collagen content was assessed using Masson's Trichome staining. Receptor abundance was assessed using RT-PCR, qRT-PCR and immunohistochemistry. In sub-epithelial stroma, there was less collagen during the follicular than luteal phase, in the caudal- (P =  0.029), mid- (P =  0.0000) and cranial (P =  0.001) cervical tissue. In the deep stroma, there was less collagen staining during the follicular stage in the mid- (P =  0.004) and cranial- (P =  0.041) cervical regions. There were PTGER2, PTGER3, PGR and ESR1 mRNA transcripts in the cervix. A greater proportion of cells were positive for ESR1 protein during the follicular phase in sub-epithelial (P =  0.019) and deep (P =  0.013) stroma. The abundance of ESR1 in the epithelium was negatively correlated with collagen staining in sub-epithelial (P =  0.007) and deep (P =  0.005) stroma. The results of the study provide new information about the cervical biology of mares by increasing the knowledge about collagen content and the relationship between collagen content and ESR1 protein abundance during the oestrous cycle which indicates the ESR1 receptor is a candidate for involvement in control of cervical dilation.


Assuntos
Colo do Útero/fisiologia , Colágeno/fisiologia , Receptor alfa de Estrogênio/metabolismo , Ciclo Estral/fisiologia , Animais , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Cavalos , Primeira Fase do Trabalho de Parto/fisiologia , Hormônio Luteinizante/genética , Hormônio Luteinizante/metabolismo , Gravidez , Progesterona/metabolismo , RNA/genética , RNA/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP3/genética , Receptores de Prostaglandina E Subtipo EP3/metabolismo
6.
ACS Appl Mater Interfaces ; 11(42): 38459-38466, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31593426

RESUMO

Upconversion nanoparticles (UCNPs) have become competitive materials for bioanalysis, bioimaging, and early diagnosis of diseases, especially cancers. However, traditional upconversion luminescence (UCL) nanosensors are often challenged with complicated covalent modification and relatively poor stability. As efficient energy acceptors in the luminescence resonance energy-transfer (LRET) process, organic dyes exhibit unique advantages such as easy modification and stable property. Herein, a simple and universal bioplatform is constructed for in situ imaging and quantitation of intracellular microRNA-21 (miR-21) using dual-acceptor-based upconversion nanoprobes with enhanced quenching efficiency. In this assay, UCNPs with core-shell structures are synthesized, in which the emitting ions are confined in the shell to take the energy donors and acceptors in close proximity. The complementary DNA (cDNA) that can specifically recognize target miR-21 is labeled with organic dyes TAMRA and black hole quencher as dual acceptors and easily assembled on UCNPs via electrostatic adsorption. Compared with only one acceptor for LRET, two dyes quench more luminescence of UCNPs (>60%), which thus reduce the background and improve the sensitivity. With the enhanced quenching efficiency and simple assembly process, the proposed system is readily applied to in situ imaging of miR-21 in different cancer cells, which further achieves quantification of miR-21 in MCF-7 cells. Therefore, our proposed dual-acceptor-based upconversion nanoplatform opens up new opportunities for sensitive analysis of miRNA and provides potential applications in biomedical and clinical research.


Assuntos
MicroRNAs/metabolismo , Nanopartículas/química , Animais , DNA Complementar/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fluoretos/química , Células HeLa , Humanos , Células MCF-7 , Camundongos , MicroRNAs/química , Microscopia Confocal , Células NIH 3T3 , Ítrio/química
7.
Elife ; 82019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31621580

RESUMO

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.


Assuntos
Aedes/virologia , Imunidade Inata , Vírus de RNA/crescimento & desenvolvimento , Vírus de RNA/imunologia , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , DNA Complementar/metabolismo , DNA Viral/metabolismo , Proteínas de Drosophila/metabolismo
8.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505762

RESUMO

Scutellaria baicalensis is a well-known medicinal plant that produces biologically active flavonoids, such as baicalin, baicalein, and wogonin. Pharmacological studies have shown that these compounds have anti-inflammatory, anti-bacterial, and anti-cancer activities. Therefore, it is of great significance to investigate the genetic information of S. baicalensis, particularly the genes related to the biosynthetic pathways of these compounds. Here, we constructed the full-length transcriptome of S. baicalensis using a hybrid sequencing strategy and acquired 338,136 full-length sequences, accounting for 93.3% of the total reads. After the removal of redundancy and correction with Illumina short reads, 75,785 nonredundant transcripts were generated, among which approximately 98% were annotated with significant hits in the protein databases, and 11,135 sequences were classified as lncRNAs. Differentially expressed gene (DEG) analysis showed that most of the genes related to flavonoid biosynthesis were highly expressed in the roots, consistent with previous reports that the flavonoids were mainly synthesized and accumulated in the roots of S. baicalensis. By constructing unique transcription models, a total of 44,071 alternative splicing (AS) events were identified, with intron retention (IR) accounting for the highest proportion (44.5%). A total of 94 AS events were present in five key genes related to flavonoid biosynthesis, suggesting that AS may play important roles in the regulation of flavonoid biosynthesis in S. baicalensis. This study provided a large number of highly accurate full-length transcripts, which represents a valuable genetic resource for further research of the molecular biology of S. baicalensis, such as the development, breeding, and biosynthesis of active ingredients.


Assuntos
DNA Complementar , Regulação da Expressão Gênica/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Raízes de Plantas , Plantas Medicinais , Scutellaria baicalensis , DNA Complementar/genética , DNA Complementar/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Scutellaria baicalensis/genética , Scutellaria baicalensis/metabolismo
9.
Mar Drugs ; 17(9)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470614

RESUMO

A very powerful proteinaceous inhibitor of metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.


Assuntos
Organismos Aquáticos/química , Proteínas/antagonistas & inibidores , Caramujos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular/métodos , DNA Complementar/metabolismo , Humanos , Especificidade por Substrato
10.
Gynecol Oncol ; 155(2): 331-339, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31493899

RESUMO

INTRODUCTION: PI3K pathway signaling has received attention as a molecular target in clear cell ovarian carcinoma (CCOC). MDM2 is one of the AKT effectors in the PI3K pathway, which binds to and degrades p53. In this study, we aimed to clarify the prognostic significance of PIK3CA and MDM2 expression, and potential therapeutic effect of a dual inhibition of the PI3K pathway and MDM2. MATERIALS AND METHODS: cDNA expression was evaluated by using microarray data using 75 samples of CCOC. DS-7423 (dual inhibitor of pan-PI3K and mTOR) and RG7112 (MDM2 inhibitor) were used on CCOC cell lines to evaluate cell proliferation, expression level of MDM2 related proteins, and apoptosis by MTT assay, western blotting, and flow cytometry. DS-7423 (3 mg/kg) and/or RG7112 (50 mg/kg) were orally administrated every day for three weeks, and the anti-tumor effect was evaluated using tumor xenografts, along with immunohistochemistry. RESULTS: Tumors with high expression of both PIK3CA and MDM2 showed significantly worse prognosis in expression array of 71 CCOCs (P = 0.013). Dual inhibition of the PI3K pathway by DS-7423 and MDM2 by RG7112 showed synergistic anti-proliferative effect in 4 CCOC cell lines without TP53 mutations. The combination therapy more robustly induced pro-apoptotic proteins (PUMA and cleaved PARP) with increase of sub G1 population and apoptotic cells, compared with either single agent alone. The combination therapy significantly reduced tumor volume in mice (P < 0.001 in OVISE, and P = 0.038 in RMG-I) without severe body weight loss. Immunohistochemistry from the xenograft tumors showed that the combination treatment significantly reduced vascularity and cell proliferation, with an increase of apoptotic cell death. CONCLUSION: A combination therapy targeting the PI3K pathway and MDM2 might be a promising therapeutic strategy in CCOC.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Adenina/análogos & derivados , Adenina/farmacologia , Adenocarcinoma de Células Claras , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA Complementar/metabolismo , Feminino , Xenoenxertos , Imidazolinas/farmacologia , Camundongos Nus , Transplante de Neoplasias/fisiologia , Neoplasias Ovarianas/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Distribuição Aleatória
11.
Curr Protoc Protein Sci ; 97(1): e88, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517450

RESUMO

Heterologous expression of the G protein-coupled estrogen receptor (GPER) comes with a suite of challenges intrinsic to membrane proteins. This receptor's low expression levels and tendency to form insoluble aggregates in Escherichia coli and yeast make it a difficult receptor-target to study. In this unit, we detail steps to produce monomeric GPER using a precipitation-based cell-free system. We provide information on the DNA construct for expression, the pipetting scheme for the reaction supplements to generate a master mix, and the cell-free reaction setup. In the last portion of this unit, we outline steps for solubilization and purification, and we provide a viable method for qualitatively observing functionality by liquid chromatography-mass spectrometry detection. © 2019 by John Wiley & Sons, Inc.


Assuntos
Escherichia coli/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/isolamento & purificação , Sistema Livre de Células/metabolismo , Cromatografia Líquida de Alta Pressão , DNA Complementar/metabolismo , Expressão Gênica , Humanos , Espectrometria de Massas em Tandem
12.
Virology ; 536: 110-118, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31419711

RESUMO

Swine enteric alphacoronavirus (SeACoV), also known as swine acute diarrhea syndrome coronavirus (SADS-CoV), belongs to the species Rhinolophus bat coronavirus HKU2. Herein, we report on the primary characterization of SeACoV in vitro. Four antibodies against the SeACoV spike, membrane, nucleocapsid and nonstructural protein 3 capable of reacting with viral antigens in SeACoV-infected Vero cells were generated. We established a DNA-launched SeACoV infectious clone based on the cell adapted passage-10 virus and rescued the recombinant virus with a unique genetic marker in cultured cells. Six subgenomic mRNAs containing the leader-body junction sites, including a bicistronic mRNA encoding the accessory NS7a and NS7b genes, were experimentally identified in SeACoV-infected cells. Cellular ultrastructural changes induced by SeACoV infection were visualized by electron microscopy. The availability of the SeACoV infectious clone and a panel of antibodies against different viral proteins will facilitate further studies on understanding the molecular mechanisms of SeACoV replication and pathogenesis.


Assuntos
Alphacoronavirus/genética , Anticorpos Antivirais/química , Antígenos Virais/química , Infecções por Coronavirus/veterinária , RNA Mensageiro/genética , RNA Viral/genética , Alphacoronavirus/metabolismo , Alphacoronavirus/patogenicidade , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Sequência de Bases , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Quirópteros , Chlorocebus aethiops , Células Clonais , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , DNA Complementar/genética , DNA Complementar/metabolismo , Microscopia Eletrônica , Nucleocapsídeo/química , Nucleocapsídeo/imunologia , RNA Replicase/química , RNA Replicase/imunologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Coelhos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/imunologia , Replicação Viral
13.
Methods Mol Biol ; 2024: 181-198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31364050

RESUMO

In the last two decades, phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Streptococcus pneumoniae-and the use of this library for the molecular dissection of the human B-cell response against bacterial infection and colonization.


Assuntos
Biblioteca de Peptídeos , Streptococcus pneumoniae/imunologia , Antígenos/imunologia , Bacteriófago lambda/metabolismo , DNA Complementar/metabolismo , Biblioteca Genômica
14.
Virology ; 536: 20-26, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31394408

RESUMO

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.


Assuntos
Adenovírus Humanos/genética , Sistemas CRISPR-Cas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Edição de Genes/métodos , Regulação Viral da Expressão Gênica , Adenovírus Humanos/metabolismo , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Cães , Doxiciclina/farmacologia , Éxons , Humanos , Células Madin Darby de Rim Canino , Regiões Promotoras Genéticas/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Guia/genética , RNA Guia/metabolismo
15.
J Biol Chem ; 294(37): 13781-13788, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31350340

RESUMO

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.


Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Transcrição Genética/genética , Sequência de Aminoácidos , Sequência de Bases , Códon/metabolismo , Códon de Iniciação/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Metabolismo Energético , Mitocôndrias/genética , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Gene ; 712: 143962, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31288057

RESUMO

Veratrum nigrum is protected plant of Melanthiaceae family, able to synthetize unique steroidal alkaloids important for pharmacy. Transcriptomes from leaves, stems and rhizomes of in vitro maintained V. nigrum plants were sequenced and annotated for genes and markers discovery. Sequencing of samples derived from the different organs resulted in a total of 108,511 contigs with a mean length of 596 bp. Transcripts derived from leaf and stalk were annotated at 28%, and 38% in Nr nucleotide database, respectively. The sequencing revealed 949 unigenes related with lipid metabolism, including 73 transcripts involved in steroids and genus-specific steroid alkaloids biosynthesis. Additionally, 3203 candidate SSRs markers we identified in unigenes with average density of one SSR locus every 6.2 kb sequence. Unraveling of biochemical machinery of the pathway responsible for steroidal alkaloids will open possibility to design and optimize biotechnological process. The transcriptomic data provide valuable resources for biochemical, molecular genetics, comparative transcriptomics, functional genomics, ecological and evolutionary studies of V. nigrum.


Assuntos
Alcaloides/biossíntese , Regulação da Expressão Gênica de Plantas , Esteroides/biossíntese , Transcriptoma , Veratrum/metabolismo , Mapeamento de Sequências Contíguas , DNA Complementar/metabolismo , Biblioteca Gênica , Ontologia Genética , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Análise de Sequência de RNA
17.
EMBO Rep ; 20(8): e47604, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31271494

RESUMO

The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF65 and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.


Assuntos
Processamento Alternativo , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Spliceossomos/genética , Fator de Processamento U2AF/genética , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , DNA Complementar/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Humanos , Motivos de Nucleotídeos , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/metabolismo , Fator de Processamento U2AF/antagonistas & inibidores , Fator de Processamento U2AF/metabolismo
18.
Plant Mol Biol ; 101(3): 257-268, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302867

RESUMO

KEY MESSAGE: The C-terminal cysteine-rich motif of NYE1/SGR1 affects chlorophyll degradation likely by mediating its self-interaction and conformational change, and somehow altering its Mg-dechelating activity in response to the changing redox potential. During green organ senescence in plants, the most prominent phenomenon is the degreening caused by net chlorophyll (Chl) loss. NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) was recently reported to be able to dechelates magnesium (Mg) from Chl a to initiate its degradation, but little is known about the domain/motif basis of its functionality. In this study, we carried out a protein truncation assay and identified a conserved cysteine-rich motif (CRM, P-X3-C-X3-C-X-C2-F-P-X5-P) at its C terminus, which is essential for its function. Genetic analysis showed that all four cysteines in the CRM were irreplaceable, and enzymatic assays demonstrated that the mutation of each of the four cysteines affected its Mg-dechelating activity. The CRM plays a critical role in the conformational change and self-interaction of NYE1 via the formation of inter- and intra-molecular disulfide bonds. Our results may provide insight into how NYE1 responds to rapid redox changes during leaf senescence and in response to various environmental stresses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Clorofila/química , Proteínas de Cloroplastos/metabolismo , Motivos de Aminoácidos , Quelantes/química , DNA Complementar/metabolismo , Dissulfetos , Regulação da Expressão Gênica de Plantas , Magnésio/química , Oxirredução , Fenótipo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Conformação Proteica , Domínios Proteicos , Estresse Fisiológico
19.
J Reprod Dev ; 65(5): 407-412, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31204365

RESUMO

Exportin 6, which functions specifically in the nuclear export of actin family proteins, has been reported to be absent in immature Xenopus oocytes, which have a huge nucleus containing a large amount of actin. In mammalian oocytes, however, the presence and the function of exportin 6 remain uninvestigated. In this study, we assessed the expression and effects of exportin 6 on meiotic resumption in porcine oocytes after cloning porcine exportin 6 cDNA and carrying out overexpression and expression inhibition by mRNA and antisense RNA injection, respectively. We found for the first time that exportin 6 was expressed in mammalian full-grown germinal-vesicle-stage oocytes and was involved in the nuclear export of actin. In contrast, exportin 6 was absent from the growing oocytes, which are meiotically incompetent and maintain the germinal-vesicle structure in the long term; the regulatory mechanism appeared to be active degradation. We examined the effects of exportin 6 on meiotic resumption of porcine oocytes and noted that its expression did not affect the onset time but increased the rate of germinal vesicle breakdown at 24 h via regulation of the nuclear actin level, which directly influences the physical strength of the germinal-vesicle membrane. Our results suggest that exportin 6 affects the nuclear transport of actin and meiotic resumption in mammalian oocytes.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Carioferinas/fisiologia , Oócitos/fisiologia , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Proliferação de Células , DNA Complementar/metabolismo , Feminino , Meiose , Oogênese , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , Suínos
20.
Protein Pept Lett ; 26(10): 785-791, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31215370

RESUMO

BACKGROUND: Laminin, a member of the Extracellular Matrix (ECM), is a glycoprotein that is used as a factor that affects cell adhesion, proliferation, survival, and differentiation. Of these, five globular domains (LG domains) of the alpha chain play an important role in influencing the cell by binding to the integrin. OBJECTIVE: This study aimed to evaluate the ability of globular domains 1-3 of laminin alpha2 (rhLAMA2LG1-3) in maintaining the pluripotency of human Mesenchymal Stem Cells (hMSCs), which are widely used in regenerative medicine. METHODS: hMSCs were grown in the medium supplemented with rhLAMA2LG1-3, then the effect of the protein on hMSCs were confirmed through cell adhesion assay, proliferation assay and RTPCR. RESULTS: rhLAMA2LG1-3 expressed in Escherichia coli has a molecular weight of 70 kDa, at 1 µg/ml concentration of rhLAMA2LG1-3, the attachment and proliferation of hMSCs were approximately 3.18-fold and 1.67-fold, respectively, more efficient than those of untreated controls. In addition, the undifferentiated state and degree of stemness of hMSCs were measured, on the basis of CD90 and CD105 levels. In the rhLAMA2LG1-3-treated hMSCs, the expression levels of CD90 and CD105 increased by 2.83-fold and 1.62-fold, respectively, compared to those in untreated controls. CONCLUSIONS: rhLAMA2LG1-3 can be potentially used in stem cell therapy to improve the viability and maintain the undifferentiated state of hMSCs.


Assuntos
Matriz Extracelular/metabolismo , Laminina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas Recombinantes/metabolismo , Adesão Celular , Proliferação de Células , Células Cultivadas , DNA Complementar/metabolismo , Escherichia coli , Humanos , Laminina/genética , Domínios Proteicos , Proteínas Recombinantes/genética
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