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1.
Parasitol Res ; 119(7): 2217-2226, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32500370

RESUMO

Schistosoma is the causative agent of schistosomiasis, a common infectious disease distributed worldwide. Our previous phosphoproteomic analysis suggested that glycogen synthase kinase 3 (GSK3), a conserved protein kinase in eukaryotes, is likely involved in protein phosphorylation of Schistosoma japonicum. Here, we aimed to identify the interacting partners of S. japonicum GSK3ß (SjGSK3ß) and to evaluate its role in parasite survival. Toward these ends, we determined the transcription levels of SjGSK3ß at different developmental stages and identified its interacting partners of SjGSK3ß by screening a yeast two-hybrid S. japonicum cDNA library. We further used RNA interference (RNAi) to inhibit the expression of SjGSK3ß in adult worms in vitro and examined the resultant changes in transcription of its putative interacting proteins and in worm viability compared with those of control worms. Reverse transcription-quantitative polymerase chain analysis indicated that SjGSK3ß is expressed throughout the life cycle of S. japonicum, with higher expression levels detected in the eggs and relatively higher expression level found in male worms than in female worms. By screening the yeast two-hybrid library, eight proteins were identified as potentially interacting with SjGSK3ß including cell division cycle 37 homolog (Cdc37), 14-3-3 protein, tegument antigen (I(H)A), V-ATPase proteolipid subunit, myosin alkali light chain 1, and three proteins without recognized functional domains. In addition, SjGSK3ß RNAi reduced the SjGSK3ß gene transcript level, leading to a significant decrease in kinase activity, cell viability, and worm survival. Collectively, these findings suggested that SjGSK3ß may interact with its partner proteins to influence worm survival by regulating kinase activity.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas de Helminto/metabolismo , Schistosoma japonicum/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Helminto/genética , Masculino , Ligação Proteica , Interferência de RNA , Schistosoma japonicum/enzimologia , Schistosoma japonicum/genética , Análise de Sobrevida , Técnicas do Sistema de Duplo-Híbrido
2.
Proc Natl Acad Sci U S A ; 117(17): 9613-9620, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284406

RESUMO

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.


Assuntos
Cloroplastos/microbiologia , Regulação da Expressão Gênica de Plantas/imunologia , Luz , Proteínas NLR/metabolismo , Phytophthora infestans/metabolismo , Proteínas de Plantas/metabolismo , Agrobacterium/metabolismo , Animais , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Inativação Gênica , Microscopia Confocal , Proteínas NLR/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Plantas/genética , Plântula , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia , Técnicas do Sistema de Duplo-Híbrido
3.
Biomed Environ Sci ; 33(3): 158-164, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32209174

RESUMO

Objective: The purpose of this study was to investigate the anti-androgenic mechanism of cypermethrin involving coactivators. Methods: Mammalian two-hybrid assays were performed to study the effects of cypermethrin on interactions of the androgen receptor (AR) with the coactivators androgen receptor-associated protein 70 (ARA70) and androgen receptor-associated protein 55 (ARA55). Results: The results showed that AR-ARA70 and AR-ARA55 interactions were remarkably enhanced by dihydrotestosterone (DHT, P ≤ 0.05). Cypermethrin inhibited DHT-induced AR-ARA70 and AR-ARA55 interactions significantly ( P ≤ 0.05). Conclusion: The study indicates that cypermethrin exhibits inhibitory effects on AR transcription associated with repression of AR-ARA70 and AR-ARA55 interactions in a ligand-dependent manner. The data show novel anti-androgenic mechanisms of cypermethrin that contribute to male reproductive toxicology.


Assuntos
Antagonistas de Androgênios/farmacologia , Inseticidas/efeitos adversos , Piretrinas/efeitos adversos , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos/efeitos adversos , Animais , Linhagem Celular , Chlorocebus aethiops , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Técnicas do Sistema de Duplo-Híbrido
4.
World J Microbiol Biotechnol ; 36(4): 56, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32211973

RESUMO

PirAB toxin was initially found in the Photorhabdus luminescens TT01 strain and is a demonstrated binary toxin with high insecticidal activity. In this paper, we co-expressed the pirAB gene of Xenorhabdus nematophila HB310 in a prokaryotic expression system, and we found that the PirAB protein showed high hemocoel insecticidal activity against Galleria mellonella, Helicoverpa armigera and Spodoptera exigua. LD50 values were 1.562, 2.003 and 2.17 µg/larvae for G. mellonella, H. armigera, and S. exigua, respectively (p > 0.05). Additionally, PirAB-interaction proteins were identified from G. mellonella by 6 × His Protein Pulldown combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of which, arylphorin of G. mellonella showed the highest matching rate. A protein domain conservative structure analysis indicated that arylphorin has three domains including Hemocyanin-N, Hemocyanin-M, and Hemocyanin-C. Among these protein domains, Hemocyanin-C has immune and recognition functions. Further, Hemocyanin-C domain of arylphorin was identified to interact with PirA but not PirB by Yeast two-hybrid system. These findings reveal, for the first time, new host protein interacting with PirAB. The identification of interaction protein may serve as the foundation for further study on the function and insecticidal mechanism of this binary toxin from Xenorhabdus.


Assuntos
Toxinas Bacterianas/farmacologia , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Xenorhabdus/metabolismo , Animais , Toxinas Bacterianas/genética , Sítios de Ligação , Cromatografia Líquida , Clonagem Molecular , Proteínas de Insetos/química , Mariposas/classificação , Mariposas/metabolismo , Ligação Proteica , Domínios Proteicos , Espectrometria de Massas em Tandem , Técnicas do Sistema de Duplo-Híbrido , Xenorhabdus/genética
5.
Parasit Vectors ; 13(1): 105, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32103780

RESUMO

BACKGROUND: Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. METHODS: The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. RESULTS: A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. CONCLUSIONS: To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.


Assuntos
Anaplasma ovis/metabolismo , Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/metabolismo , Dermacentor/metabolismo , Dermacentor/microbiologia , Sistemas de Secreção Tipo IV/metabolismo , Anaplasma ovis/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Bactérias/genética , Dermacentor/genética , Interações Hospedeiro-Parasita , Ligação Proteica , Glândulas Salivares/metabolismo , Glândulas Salivares/microbiologia , Técnicas do Sistema de Duplo-Híbrido , Sistemas de Secreção Tipo IV/genética
6.
Virology ; 542: 54-62, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32056668

RESUMO

Intergenic region of begomovirus genome is vital to virus replication and viral gene transcription in plants. Previous studies have reported that Tomato yellow leaf curl China virus (TYLCCNV), a begomovirus, is able to accumulate and transcribe in its whitefly vector. However, the viral and host components that participate in begomovirus transcription in whiteflies are hitherto unknown. Using a yeast one-hybrid system, we identified >50 whitefly proteins that interacted with TYLCCNV intergenic region. Dual luciferase analysis revealed that one of the identified proteins, the hairy and enhancer of split homolog-1 (HES1), specifically bound to CACGTG motif in TYLCCNV intergenic region. Silencing HES1 decreased viral transcription, accumulation and transmission. These results demonstrate that the interactions between whitefly proteins and the intergenic region of TYLCCNV may contribute to viral transcription in the whitefly vector. Our findings offer valuable clues for the research and development of novel strategies to interfere with begomovirus transmission.


Assuntos
Begomovirus/genética , Hemípteros/metabolismo , Hemípteros/virologia , Proteínas de Insetos/metabolismo , Fatores de Transcrição HES-1/metabolismo , Animais , Begomovirus/patogenicidade , Begomovirus/fisiologia , DNA Intergênico , Técnicas de Silenciamento de Genes , Genoma Viral , Hemípteros/genética , Interações entre Hospedeiro e Microrganismos/genética , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Insetos Vetores/genética , Insetos Vetores/metabolismo , Insetos Vetores/virologia , Lycopersicon esculentum/virologia , Doenças das Plantas/virologia , Ligação Proteica , Tabaco/virologia , Fatores de Transcrição HES-1/antagonistas & inibidores , Fatores de Transcrição HES-1/genética , Transcrição Genética , Técnicas do Sistema de Duplo-Híbrido
7.
BMC Res Notes ; 12(1): 804, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900205

RESUMO

OBJECTIVES: Family with sequence similarity 13 member A (FAM13A) genetic variants have been associated with several chronic respiratory diseases including chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF) and lung cancer. The FAM13A protein includes a RhoGTPase activating protein (RhoGAP) domain known to participate in various cellular mechanisms including cell proliferation. While intensive genomic studies have been performed to reveal its involvement in lung diseases, the biological role of FAM13A protein is still not completely elucidated. RESULTS: We therefore performed a two-hybrid screening to identify protein partners of FAM13A using a human lung cancer cDNA library. We identified several protein partners with a high confidence score. Researchers in the field of chronic lung diseases may benefit from this two-hybrid screening data which may reveal new research pathways to decipher.


Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Células A549 , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/genética , Biblioteca Gênica , Humanos , Pulmão/citologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Ligação Proteica
8.
Mol Cell Biol ; 40(7)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-31932482

RESUMO

TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Infertilidade Masculina/genética , Espermatogênese/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovário/metabolismo , Protaminas/genética , Protaminas/metabolismo , Domínios Proteicos/genética , Testículo/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional/genética , Técnicas do Sistema de Duplo-Híbrido
9.
Plant Cell Rep ; 39(4): 489-500, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31900582

RESUMO

KEY MESSAGE: OsNINJA1-interacting protein, OsSRO1a, acts as a mediator that suppresses OsMYC2 activity in response to JA. Jasmonic acid (JA) is an important plant hormone for the stable growth and development of higher plants. The rice gene NOVEL INTERACTOR OF JAZ1 (OsNINJA1) interacts with Jasmonate ZIM-domain (JAZ) proteins and is a repressor of JA signaling. In this study, we identified several OsNINJA1-interacting proteins in rice from a yeast two-hybrid screen. Among the newly identified genes, we focused on SIMILAR TO RCD ONE1a (OsSRO1a) and investigated its role in JA signaling. Full-length OsSRO1a interacted with OsNINJA1 in plant cells but not in yeast cells. OsSRO1a also interacted with OsMYC2, a positive transcription factor in JA signaling, in both plant and yeast cells. The expression of OsSRO1a was upregulated at a late phase after JA treatment. Transgenic rice plants overexpressing OsSRO1a exhibited JA-insensitive phenotypes. In wild-type plants, JA induces resistance against rice bacterial blight, but this phenotype was suppressed in the OsSRO1a-overexpressing plants. Furthermore, the degradation of chlorophyll under dark-induced senescence conditions and the JA-induced upregulation of OsMYC2-responsive genes were suppressed in the OsSRO1a-overexpressing plants. These results suggest that OsSRO1a is a negative regulator of the OsMYC2-mediated JA signaling pathway in rice.


Assuntos
Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Oryza/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/genética , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Oryza/genética , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/efeitos da radiação , Técnicas do Sistema de Duplo-Híbrido , Regulação para Cima
10.
Biochim Biophys Acta Mol Cell Res ; 1867(2): 118611, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31751593

RESUMO

Methylation of proteins is emerging to be an important regulator of protein function. SET7/9, a protein lysine methyltransferase, catalyses methylation of several proteins involved in diverse biological processes. SET7/9-mediated methylation often regulates the stability, sub-cellular localization and protein-protein interactions of its substrate proteins. Here, we aimed to identify novel biological processes regulated by SET7/9 by identifying new interaction partners. For this we used yeast two-hybrid screening and identified the large subunit ribosomal protein, eL42 as a potential interactor of SET7/9. We confirmed the SET7/9-eL42 interaction by co-immunoprecipitation and GST pulldown studies. The N-terminal MORN domain of SET7/9 is essential for its interaction with eL42. Importantly, we identified that SET7/9 methylates eL42 at three different lysines - Lys53, Lys80 and Lys100 through site-directed mutagenesis. By puromycin incorporation assay, we find that SET7/9-mediated methylation of eL42 affects global translation. This study identifies a new role of the functionally versatile SET7/9 lysine methyltransferase in the regulation of global protein synthesis.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Células HEK293 , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Humanos , Lisina/química , Metilação , Biossíntese de Proteínas , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Técnicas do Sistema de Duplo-Híbrido
11.
Dev Biol ; 457(1): 91-103, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31550482

RESUMO

Little is known about the role of TBX1 in post-otocyst stages of inner ear development. Here, we report on mice with a missense mutation of Tbx1 that are viable with fully developed but abnormally formed inner ears. Mutant mice are deaf due to an undeveloped stria vascularis and show vestibular dysfunction associated with abnormal semicircular canal formation. We show that TBX1 is expressed in endolymph-producing strial marginal cells and vestibular dark cells of the inner ear and is an upstream regulator of Esrrb, which previously was shown to control the developmental fate of these cells. We also show that TBX1 is expressed in sensory cells of the crista ampullaris, which may relate to the semicircular canal abnormalities observed in mutant mice. Inner ears of mutant embryos have a non-resorbed fusion plate in the posterior semicircular canal and a single ampulla connecting anterior and lateral canals. We hypothesize that the TBX1 missense mutation prevents binding with specific co-regulatory proteins. These findings reveal previously unknown functions of TBX1 during later stages of inner ear development.


Assuntos
Orelha Interna/embriologia , Mutação de Sentido Incorreto , Canais Semicirculares/embriologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Orelha Interna/citologia , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Morfogênese , Receptores Estrogênicos/metabolismo , Canais Semicirculares/anormalidades , Estria Vascular/citologia , Proteínas com Domínio T/química , Técnicas do Sistema de Duplo-Híbrido , Sequenciamento Completo do Exoma
12.
Parasit Vectors ; 12(1): 568, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783771

RESUMO

BACKGROUND: Giardia duodenalis causes giardiasis, with diarrhea as the primary symptom. The trophozoite proliferation of this zoonotic parasite is mainly affected by telomerase, although the mechanism of telomerase regulation has not been thoroughly analyzed. METHODS: This study was performed to identify the telomerase RNA-binding domain (TRBD)-interacting protein in G. duodenalis and its regulation of telomerase. Interaction between TRBD and interacting proteins was verified via pulldown assays and co-immunoprecipitation (co-IP) techniques, and the subcellular localization of the protein interactions was determined in vivo via split SNAP-tag labeling. The hammerhead ribozyme was designed to deplete the mRNA of TRBD-interacting proteins. RESULTS: Using TRBD as bait, we identified zinc-finger domain (ZFD)-containing proteins and verified it via pulldown and co-IP experiments. Protein-protein interaction occurred in the nuclei of 293T cells and both nuclei of G. duodenalis. The hammerhead ribozyme depleted ZFD mRNA levels, which reduced the reproduction rate of G. duodenalis, telomerase activity and telomere length. CONCLUSIONS: Our findings suggest that ZFD may regulate telomere function in G. duodenalis nuclei.


Assuntos
Regulação da Expressão Gênica , Giardia lamblia/genética , Proteínas de Protozoários/metabolismo , Telomerase/genética , Dedos de Zinco , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Proteínas de Protozoários/genética , RNA/genética , RNA Catalítico/metabolismo , Telomerase/metabolismo , Técnicas do Sistema de Duplo-Híbrido
13.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801204

RESUMO

The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the jasmonic acid (JA) signaling pathways of plants, and these proteins have been reported to play key roles in plant secondary metabolism mediated by JA. In this study, we firstly isolated one JAZ from P. cablin, PatJAZ6, which was characterized and revealed based on multiple alignments and a phylogenic tree analysis. The result of subcellular localization indicated that the PatJAZ6 protein was located in the nucleus of plant protoplasts. The expression level of PatJAZ6 was significantly induced by the methyl jasmonate (MeJA). Furthermore, by means of yeast two-hybrid screening, we identified two transcription factors that interact with the PatJAZ6, the PatMYC2b1 and PatMYC2b2. Virus-induced gene silencing (VIGS) of PatJAZ6 caused a decrease in expression abundance, resulting in a significant increase in the accumulation of patchouli alcohol. Moreover, we overexpressed PatJAZ6 in P. cablin, which down-regulated the patchoulol synthase expression, and then suppressed the biosynthesis of patchouli alcohol. The results demonstrate that PatJAZ6 probably acts as a repressor in the regulation of patchouli alcohol biosynthesis, contributed to a model proposed for the potential JA signaling pathway in P. cablin.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Pogostemon/genética , Proteínas Repressoras/genética , Sesquiterpenos/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Inativação Gênica , Isomerases/genética , Isomerases/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Filogenia , Reguladores de Crescimento de Planta/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Pogostemon/classificação , Pogostemon/efeitos dos fármacos , Pogostemon/metabolismo , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Técnicas do Sistema de Duplo-Híbrido
14.
PLoS Genet ; 15(12): e1008508, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31815936

RESUMO

Zinc is essential for cellular functions as it is a catalytic and structural component of many proteins. In contrast, cadmium is not required in biological systems and is toxic. Zinc and cadmium levels are closely monitored and regulated as their excess causes cell stress. To maintain homeostasis, organisms induce metal detoxification gene programs through stress responsive transcriptional regulatory complexes. In Caenorhabditis elegans, the MDT-15 subunit of the evolutionarily conserved Mediator transcriptional coregulator is required to induce genes upon exposure to excess zinc and cadmium. However, the regulatory partners of MDT-15 in this response, its role in cellular and physiological stress adaptation, and the putative role for mammalian MED15 in the metal stress responses remain unknown. Here, we show that MDT-15 interacts physically and functionally with the Nuclear Hormone Receptor HIZR-1 to promote molecular, cellular, and organismal adaptation to cadmium and excess zinc. Using gain- and loss-of-function mutants and qRT-PCR and reporter analysis, we find that mdt-15 and hizr-1 cooperate to induce zinc and cadmium responsive genes. Moreover, the two proteins interact physically in yeast-two-hybrid assays and this interaction is enhanced by the addition of zinc or cadmium, the former a known ligand of HIZR-1. Functionally, mdt-15 and hizr-1 mutants show defective storage of excess zinc in the gut and are hypersensitive to zinc-induced reductions in egg-laying. Furthermore, mdt-15 but not hizr-1 mutants are hypersensitive to cadmium-induced reductions in egg-laying, suggesting potential divergence of regulatory pathways. Lastly, mammalian MDT-15 orthologs bind genomic regulatory regions of metallothionein and zinc transporter genes in a cadmium and zinc-stimulated fashion, and human MED15 is required to induce a metallothionein gene in lung adenocarcinoma cells exposed to cadmium. Collectively, our data show that mdt-15 and hizr-1 cooperate to regulate cadmium detoxification and zinc storage and that this mechanism is at least partially conserved in mammals.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Zinco/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Humanos , Metalotioneína/genética , Mutação , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Estresse Fisiológico , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
15.
Int J Mol Sci ; 21(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878192

RESUMO

Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3-ATF4 (not hRPB11a-ATF4) and hRpb11bα-ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3-Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11-ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Isoformas de Proteínas/metabolismo , RNA Polimerase II/metabolismo , Fator 4 Ativador da Transcrição/genética , Humanos , Ligação Proteica , Isoformas de Proteínas/genética , RNA Polimerase II/genética , Técnicas do Sistema de Duplo-Híbrido
16.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 1000-1007, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31878996

RESUMO

Objective To screen a standard homogenous cDNA library of human gastric mucosal epithelium with yeast two-hybrid system and find the proteins that interact with Src-homology 2-containing inositol 5-phosphatase 2 (SHIP2). Methods Using the yeast two-hybrid system, P1 (SH2+5-Ptase) and P2 (PRD+SAM) segments of SHIP2 were used as bait proteins to screen the proteins that bind to SHIP2 from the homogenous cDNA library of human gastric mucosal epithelium. The selected interacting proteins of SHIP2 were verified by co-immunoprecipitation assay. Results A total of 39 positive clones were selected and sequenced for alignment analysis. It was verified that PHB interacted with SHIP2 by reductive hybridization and co-immunoprecipitation assays. Conclusion PHB, the interacting protein of SHIP2 was screened by yeast two-hybrid system from the homogenous cDNA library of human gastric mucosal epithelium.


Assuntos
Epitélio/metabolismo , Mucosa Gástrica/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Repressoras/metabolismo , Biblioteca Gênica , Humanos , Inositol Polifosfato 5-Fosfatases , Técnicas do Sistema de Duplo-Híbrido
17.
Viruses ; 11(12)2019 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-31847316

RESUMO

Host proteins that are central to infection of potyviruses (genus Potyvirus; family Potyviridae) include the eukaryotic translation initiation factors eIF4E and eIF(iso)4E. The potyviral genome-linked protein (VPg) and the helper component proteinase (HCpro) interact with each other and with eIF4E and eIF(iso)4E and proteins are involved in the same functions during viral infection. VPg interacts with eIF4E/eIF(iso)4E via the 7-methylguanosine cap-binding region, whereas HCpro interacts with eIF4E/eIF(iso)4E via the 4E-binding motif YXXXXLΦ, similar to the motif in eIF4G. In this study, HCpro and VPg were found to interact in the nucleus, nucleolus, and cytoplasm in cells infected with the potyvirus potato virus A (PVA). In the cytoplasm, interactions between HCpro and VPg occurred in punctate bodies not associated with viral replication vesicles. In addition to HCpro, the 4E-binding motif was recognized in VPg of PVA. Mutations in the 4E-binding motif of VPg from PVA weakened interactions with eIF4E and heavily reduced PVA virulence. Furthermore, mutations in the 4G-binding domain of eIF4E reduced interactions with VPg and abolished interactions with HCpro. Thus, HCpro and VPg can both interact with eIF4E using the 4E-binding motif. Our results suggest a novel interaction network used by potyviruses to interact with host plants via translation initiation factors.


Assuntos
Interações Hospedeiro-Patógeno , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Potyvirus/fisiologia , Mapas de Interação de Proteínas , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Núcleo Celular , Mutação , Fenótipo , Proteínas de Plantas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Transporte Proteico , Tabaco/virologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Replicação Viral
18.
Parasitol Res ; 118(12): 3387-3398, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31728719

RESUMO

Leucine aminopeptidase of Taenia pisiformis (TpLAP) belonging to the M17 peptidase family has been implicated as a stage-differentially expressed protein in the adult stage of T. pisiformis. In order to further dissect the biological functions of TpLAP in the growth and development of adult worms, TpLAP-interacting partners were investigated. In this study, a yeast two-hybrid (Y2H) cDNA library from adult T. pisiformis was constructed. Using pGBKT7-TpLAP as bait, proteins interacting with TpLAP were screened by Y2H system and positive preys were sequenced and analyzed using the Basic Local Alignment Search Tool (BLAST). Our results showed that six genuine TpLAP-interacting proteins, including LAP, dynein light chain (DLC), SUMO-conjugating enzyme (UBC9), histone-lysine n-methyltransferase, trans-acting transcriptional, and one unknown protein, were identified via Y2H assay. Furthermore, the interaction between TpLAP and UBC9 of T. pisiformis (TpUBC9), an important protein involved in SUMOylation pathway, was further validated by one-to-one Y2H assay, co-immunoprecipitation, and confocal analysis. These findings provide a deeper understanding of the biological functions of TpLAP and offer the first clue that TpLAP may act as a novel SUMOylated substrate, suggesting that the SUMO modification pathway plays an important role in regulation of adult worm growth and development.


Assuntos
Leucil Aminopeptidase/metabolismo , Proteínas de Protozoários/metabolismo , Taenia/metabolismo , Animais , Sequência de Bases , Dineínas/metabolismo , Biblioteca Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imunoprecipitação , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/metabolismo
19.
PLoS One ; 14(11): e0225782, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31770407

RESUMO

Interleukin (IL)-38 is a member of the IL-1 family of cytokines, which was proposed to exert anti-inflammatory effects. IL-38 is constitutively expressed in the skin, where keratinocytes are the main producing cells. Little information is currently available concerning IL-38 biology. Here, we investigated the subcellular localization and interaction partners of the IL-38 protein in human keratinocytes. IL-38 expression was reduced in primary keratinocytes grown in monolayer (2D) cultures. We thus used IL-38 overexpressing immortalized normal human keratinocytes (NHK/38) to study this cytokine in cell monolayers. In parallel, differentiation of primary human keratinocytes in an in vitro reconstructed human epidermis (RHE) 3D model allowed us to restore endogenous IL-38 expression. In NHK/38 cells and in RHE, IL-38 was mainly cell-associated, rather than released into culture supernatants. Intracellular IL-38 was preferentially, although not exclusively, cytoplasmic. Similarly, in normal human skin sections, IL-38 was predominantly cytoplasmic in the epidermis and essentially excluded from keratinocyte nuclei. A yeast two-hybrid screen identified destrin/actin-depolymerizing factor (DSTN) as a potential IL-38-interacting molecule. Co-immunoprecipitation and proximity ligation assay confirmed this interaction. We further observed partial co-localization of IL-38 and DSTN in NHK/38 cells. Endogenous IL-38 and DSTN were also co-expressed in all epidermal layers in RHE and in normal human skin. Finally, IL-38 partially co-localized with F-actin in NHK/38 cells, in particular along the cortical actin network and in filopodia. In conclusion, IL-38 is found predominantly in the cytoplasm of human keratinocytes, where it interacts with DSTN. The functional relevance of this interaction remains to be investigated.


Assuntos
Destrina/metabolismo , Interleucinas/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Destrina/química , Humanos , Interleucinas/genética , Queratinócitos/citologia , Queratinócitos/metabolismo , Microscopia de Fluorescência , Ligação Proteica , Pele/citologia , Técnicas do Sistema de Duplo-Híbrido
20.
Int J Mol Sci ; 20(23)2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31771139

RESUMO

Deleterious mutations in Breast Cancer 1 (BRCA1) are associated with an increased risk of breast and ovarian cancer. Mutations in the tandem BRCA1 C-terminal (tBRCT) protein domain disrupt critical protein interactions required for the faithful repair of DNA through homologous recombination, which contributes to oncogenesis. Our studies have identified RICTOR, PRR5, and SIN1 subunits of the mammalian target of rapamycin complex 2 (mTORC2) as interacting partners with the tBRCT domain of BRCA1 leading to the disruption of the mTORC2 complex. However, the interplay between mTORC2 signaling and BRCA1 function in the DNA damage response (DDR) remains to be determined. In this study, we used protein interaction assays to determine the binary interactions between the tBRCT domain and mTORC2 subunits, evaluated the impact of mTOR inhibition on the transcriptional function of the tBRCT, evaluated the impact of mTOR signaling on BRCA1 recruitment to DNA damage-induced foci and determined the breast cancer cell line response to mTOR inhibition dependent upon BRCA1 expression and mutation. This study determined that PRR5, RICTOR, and SIN1 could each independently interact with the BRCA1 tBRCT. Inhibition of mTORC1, but not mTORC1/2, increases BRCA1 transcriptional activation activity. Treatment with pan-mTOR inhibitor PP242 diminishes DNA damage-induced γH2AX and BRCA1 foci formation. Breast cancer cells lacking expression of functional BRCA1 are more sensitive to mTOR inhibitors. These data suggest that mTOR signaling is required for BRCA1 response to DNA damage and breast cancer cells lacking BRCA1 are more sensitive to pan-mTOR inhibition. This work suggests chemotherapeutic strategies using mTOR inhibitors could be tailored for patients that lack functional BRCA1.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteína BRCA1/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA/genética , Dano ao DNA/fisiologia , Imunofluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Fosforilação/genética , Fosforilação/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-Híbrido
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