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1.
Curr Protoc ; 1(7): e190, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34260831

RESUMO

Protein-protein interactions (PPIs) are ubiquitously involved in cellular processes such as gene expression, enzymatic catalysis, and signal transduction. To study dynamic PPIs, real-time methods such as Förster resonance energy transfer and bioluminescence resonance energy transfer can provide high temporal resolution, but they only allow PPI detection in a limited area at a time and do not permit post-PPI analysis or manipulation of the cells. Integration methods such as the yeast two-hybrid system and split protein systems integrate PPI signals over time and allow subsequent analysis, but they lose information on dynamics. To address some of these limitations, an assay named SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics) has recently been published. Similar to many existing integrators, SPARK converts PPIs into a transcriptional signal. SPARK, however, also adds blue light as a co-stimulus to achieve temporal gating; SPARK only records PPIs during light stimulation. Here, we describe the procedures for using SPARK assays to study a dynamic PPI of interest, including designing DNA constructs and optimization in HEK293T/17 cell cultures. These protocols are generally applicable to various PPI partners and can be used in different biological contexts. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Designing DNA constructs for SPARK Basic Protocol 2: Performing the SPARK assay in HEK293T/17 cell cultures Support Protocol 1: Lentivirus preparation Support Protocol 2: Immunostaining of SPARK components.


Assuntos
Mapeamento de Interação de Proteínas , Proteínas , Células HEK293 , Humanos , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(7): 631-634, 2021 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-34247365

RESUMO

OBJECTIVE: To screen proteins interacting with ring finger protein 216(RNF216) through yeast two hybrid experiment, and further clarify the role of RNF216 in the pathogenesis of gonadotropin-releasing hormone deficiency. METHODS: A recombinant expression vector pGBKT7-RNF216 was constructed and transformed into yeast Y2HGold, which was hybridized with a human cDNA library in order to screen proteins interacting with RNF216. The interaction was verified in yeast Y2HGold. RESULTS: A recombinant expression vector pGBKT7-RNF216 was successfully constructed and expressed in yeast Y2HGold. Filamin B (FLNB) was identified by yeast two hybrid experiment, and their interaction was verified in yeast Y2HGold. CONCLUSION: An interaction between FLNB and RNF216 was identified through yeast two hybrid experiment. RNF216 may affect the proliferation and migration of GnRH neurons by regulating FLNB or FLNB/FLNA heterodimers.


Assuntos
Hormônio Liberador de Gonadotropina , Proteínas , Biblioteca Gênica , Hormônio Liberador de Gonadotropina/genética , Humanos , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética
3.
Ecotoxicol Environ Saf ; 221: 112469, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34198190

RESUMO

Glutathione S-transferase (GST) is the key enzyme in glutathione (GSH) synthesis, and plays a crucial role in copper (Cu) detoxification. Nonetheless, its regulatory mechanisms remain largely unclear. In this study, we identified a Cu-induced glutathione S-transferase 1 (TaGST1) gene in wheat. Yeast one-hybrid (Y1H) screened out TaWRKY74, which was one member from the WRKY transcription factor family. The bindings between TaGST1 promoter and TaWRKY74 were further verified by using another Y1H and luciferase assays. Expression of TaWRKY74 was induced more than 30-folds by Cu stress. Functions of TaWRKY74 were tested by using transiently silence methods. In transiently TaWRKY74-silenced wheat plants, TaWRKY74 and TaGST1 expression, GST activity, and GSH content was significantly inhibited by 25.68%, 19.88%, 27.66%, and 12.68% in shoots, and 53.81%, 52.11%, 23.47%, and 17.11% in roots, respectively. However, contents of hydrogen peroxide, malondialdehyde, or Cu were significantly increased by 2.58%, 12.45%, or 37.74% in shoots, and 25.24%, 53.84%, and 103.99% in roots, respectively. Notably, exogenous application of GSH reversed the adverse effects of transiently TaWRKY74-silenced wheat plants during Cu stress. Taken together, our results suggesting that TaWRKY74 regulated TaGST1 expression and affected GSH accumulation under Cu stress, and could be useful to ameliorate Cu toxicity for crop food safety.


Assuntos
Cobre/toxicidade , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Triticum/efeitos dos fármacos , Fatores de Transcrição/genética , Triticum/genética , Triticum/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genética
4.
Molecules ; 26(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206124

RESUMO

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.


Assuntos
Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/biossíntese , Ciclização , Humanos , Biblioteca de Peptídeos , Técnicas do Sistema de Duplo-Híbrido
5.
BMC Plant Biol ; 21(1): 284, 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34157974

RESUMO

BACKGROUND: Identifying genes involved in salt tolerance in the recretohalophyte Limonium bicolor could facilitate the breeding of crops with enhanced salt tolerance. Here we cloned the previously uncharacterized gene LbHLH and explored its role in salt tolerance. RESULTS: The 2,067-bp open reading frame of LbHLH encodes a 688-amino-acid protein with a typical helix-loop-helix (HLH) domain. In situ hybridization showed that LbHLH is expressed in salt glands of L. bicolor. LbHLH localizes to the nucleus, and LbHLH is highly expressed during salt gland development and in response to NaCl treatment. To further explore its function, we heterologously expressed LbHLH in Arabidopsis thaliana under the 35S promoter. The overexpression lines showed significantly increased trichome number and reduced root hair number. LbHLH might interact with GLABRA1 to influence trichome and root hair development, as revealed by yeast two-hybrid analysis. The transgenic lines showed higher germination percentages and longer roots than the wild type under NaCl treatment. Analysis of seedlings grown on medium containing sorbitol with the same osmotic pressure as 100 mM NaCl demonstrated that overexpressing LbHLH enhanced osmotic resistance. CONCLUSION: These results indicate that LbHLH enhances salt tolerance by reducing root hair development and enhancing osmotic resistance under NaCl stress.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plumbaginaceae/genética , Plantas Tolerantes a Sal/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Clonagem Molecular , Genes de Plantas/fisiologia , Hibridização In Situ , Pressão Osmótica , Proteínas de Plantas/fisiologia , Plumbaginaceae/metabolismo , Plumbaginaceae/fisiologia , Reação em Cadeia da Polimerase , Estresse Salino , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/metabolismo , Plantas Tolerantes a Sal/fisiologia , Técnicas do Sistema de Duplo-Híbrido
6.
Parasitol Res ; 120(7): 2617-2629, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34142223

RESUMO

Proteins containing WD40 domains play important roles in the formation of multiprotein complexes. Little is known about WD40 proteins in the malaria parasite. This report contains the initial description of a WD40 protein that is unique to the genus Plasmodium and possibly closely related genera. The N-terminal portion of this protein consists of seven WD40 repeats that are highly conserved in all Plasmodium species. Following the N-terminal region is a central region that is conserved within the major Plasmodium clades, such as parasites of great apes, monkeys, rodents, and birds, but partially conserved across all Plasmodium species. This central region contains extensive low-complexity sequence and is predicted to have a disordered structure. Proteins with disordered structure generally function in molecular interactions. The C-terminal region is semi-conserved across all Plasmodium species and has no notable features. This WD40 repeat protein likely functions in some aspect of parasite biology that is unique to Plasmodium and this uniqueness makes the protein a possible target for therapeutic intervention.


Assuntos
Plasmodium/genética , Proteínas de Protozoários/isolamento & purificação , Repetições WD40 , Sequência de Aminoácidos , Animais , Aves , Clonagem Molecular , Epitopos/química , Regulação da Expressão Gênica , Modelos Químicos , Parasitos/metabolismo , Peptídeo Hidrolases/química , Plasmodium/classificação , Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Técnicas do Sistema de Duplo-Híbrido
7.
Plant Cell Rep ; 40(7): 1269-1284, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34052884

RESUMO

KEY MESSAGE: Herein, 37 ARF genes were identified and analyzed in Hedychium coronarium and HcARF5 showed a potential role in the regulation of HcTPS3. Auxin is an important plant hormone, implicated in various aspects of plant growth and development processes especially in the biosynthesis of various secondary metabolites. Auxin response factors (ARF) belong to the transcription factors (TFs) gene family and play a crucial role in transcriptional activation/repression of auxin-responsive genes by directly binding to their promoter region. Nevertheless, whether ARF genes are involved in the regulatory mechanism of volatile compounds in flowering plants is largely unknown. ß-ocimene is a key floral volatile compound synthesized by terpene synthase 3 (HcTPS3) in Hedychium coronarium. A comprehensive analysis of H. coronarium genome reveals 37 candidate ARF genes in the whole genome. Tissue-specific expression patterns of HcARFs family members were assessed using available transcriptome data. Among them, HcARF5 showed a higher expression level in flowers, and significantly correlated with the key structural ß-ocimene synthesis gene (HcTPS3). Furthermore, transcript levels of both genes were associated with the flower development. Under hormone treatments, the response of HcARF5 and HcTPS3, and the emission level of ß-ocimene contents were evaluated. Subcellular and transcriptional activity assay showed that HcARF5 localizes to the nucleus and possesses transcriptional activity. Yeast one-hybrid (Y1H) and dual-luciferase assays revealed that HcARF5 directly regulates the transcriptional activity of HcTPS3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that HcARF5 interacts with scent-related HcIAA4, HcIAA6, and HcMYB1 in vivo. Overall, these results indicate that HcARF5 is potentially involved in the regulation of ß-ocimene synthesis in H. coronarium.


Assuntos
Monoterpenos Acíclicos/metabolismo , Alcenos/metabolismo , Alquil e Aril Transferases/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Zingiberaceae/genética , Alquil e Aril Transferases/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , MicroRNAs , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Zingiberaceae/efeitos dos fármacos , Zingiberaceae/metabolismo
8.
J Plant Physiol ; 262: 153439, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34023806

RESUMO

Late stages of floret development, such as booting, heading, and anthesis stages, are important steps for determining grain setting and for filling in wheat. Herein, we report the molecular function of Triticum aestivum ELF7 encoding RNA polymerase II-associated factor 1 (PAF1), which may act as a negative regulator in floret development and anthesis stages. Among the six TaELF7-like genes isolated from wheat, TaELF7 like1-A and TaELF7 like2-B showed contrasting expression levels during the late stage of floret development stages, with observation of decreased expression level of TaELF7 like1-A compared to that of TaELF7 like2-B. The full-length TaELF7 like1-A has a 1038-bp open reading frame that contains a proline-rich domain in the N-terminal region and a nuclear localization signal domain in the C-terminal region. TaELF7 like1-A was found to be localized in the nucleus in both tobacco and wheat. Direct interaction of TaELF7 with the RING-type E3 ligase TaHUB2 was confirmed using a yeast two-hybrid system, an in vitro pull-down assay, and a bimolecular fluorescence complementation assay. The flowering time was delayed in TaELF7-overexpressing plants compared to that in the control plants. Expression levels of few floral repressor genes were markedly increased in TaELF7-overexpressing Arabidopsis plants.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Triticum/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Técnicas do Sistema de Duplo-Híbrido
9.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946798

RESUMO

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.


Assuntos
Reações Antígeno-Anticorpo , Proteínas Imobilizadas/imunologia , Receptores Acoplados a Proteínas G/imunologia , Anticorpos de Cadeia Única/imunologia , Técnicas do Sistema de Duplo-Híbrido , Anticorpos Imobilizados/imunologia , Colorimetria , Proteínas de Ligação a DNA , Epitopos/imunologia , Genes Reporter , Humanos , Proteínas de Membrana , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR4/imunologia , Receptores CXCR5/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Ubiquitina/genética
10.
Methods Mol Biol ; 2256: 1-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34014513

RESUMO

The yeast two-hybrid technique is a powerful method to detect direct protein-protein interactions. Due to its accessibility, speed, and versatility, this technique is easy to set up in any laboratory and suitable for small and large scale screenings. Here we describe the implementation of an array-based screening that allows for the probing of the entire human PDZ ORFeome (or hPDZome) by yeast two-hybrid technique. With this approach, one can rapidly identify the PDZ domains that are able to interact (up to KD in the high µmolar range) with any candidate protein among a panel of 266 individual clones, thereby comprehensively identifying its PDZ interactome.


Assuntos
Domínios PDZ , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Humanos , Ligação Proteica
11.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33827941

RESUMO

The classical swine fever virus (CSFV) glycoprotein E2 is the major structural component of the virus particle. E2 is involved in several functions, such as virus adsorption to the cell, the elicitation of protective immune responses, and virus virulence in swine. Using a yeast two-hybrid system, we previously identified the swine host protein Torsin-1A, an ATPase protein residing in the endoplasmic reticulum and inner nucleus membrane of the cell, as a specific binding partner for E2. The interaction between Torsin-1A and E2 proteins was confirmed to occur in CSFV-infected swine cells using three independent methods: coimmunoprecipitation, confocal microscopy, and proximity ligation assay (PLA). Furthermore, the E2 residue critical to mediate the protein-protein interaction with Torsin-1A was identified by a reverse yeast two-hybrid assay using a randomly mutated E2 library. A recombinant CSFV E2 mutant protein with a Q316L substitution failed to bind swine Torsin-1A in the yeast two-hybrid model. In addition, a CSFV infectious clone harboring the E2 Q316L substitution, although expressing substantial levels of E2 protein, repetitively failed to produce virus progeny when the corresponding RNA was transfected into susceptible SK6 cells. Importantly, PLA analysis of the transfected cells demonstrated an abolishment of the interaction between E2 Q316L and Torsin-1A, indicating a critical role for that interaction during CSFV replication.IMPORTANCE Structural glycoprotein E2 is an important structural component of the CSFV particle. E2 is involved in several virus functions, particularly virus-host interactions. Here, we characterized the interaction between CSFV E2 and swine protein Torsin-1A during virus infection. The critical amino acid residue in E2 mediating the interaction with Torsin-1A was identified and the effect of disrupting the E2-Torsin-1A protein-protein interaction was studied using reverse genetics. It is shown that the amino acid substitution abrogating E2-Torsin-1A interaction constitutes a lethal mutation, demonstrating that this virus-host protein-protein interaction is a critical factor during CSFV replication. This highlights the potential importance of the E2-Torsin-1A protein-protein interaction during CSFV replication and provides a potential pathway toward blocking virus replication, an important step toward the potential development of novel virus countermeasures.


Assuntos
Vírus da Febre Suína Clássica/fisiologia , Chaperonas Moleculares/metabolismo , Proteínas do Envelope Viral/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Vírus da Febre Suína Clássica/metabolismo , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Proteínas Recombinantes/metabolismo , Suínos , Técnicas do Sistema de Duplo-Híbrido , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Replicação Viral
12.
Sci Data ; 8(1): 100, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846359

RESUMO

Progesterone receptor (PR) isoforms, PRA and PRB, act in a progesterone-independent and dependent manner to differentially modulate the biology of breast cancer cells. Here we show that the differences in PRA and PRB structure facilitate the binding of common and distinct protein interacting partners affecting the downstream signaling events of each PR-isoform. Tet-inducible HA-tagged PRA or HA-tagged PRB constructs were expressed in T47DC42 (PR/ER negative) breast cancer cells. Affinity purification coupled with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry technique was performed to comprehensively study PRA and PRB interacting partners in both unliganded and liganded conditions. To validate our findings, we applied both forward and reverse SILAC conditions to effectively minimize experimental errors. These datasets will be useful in investigating PRA- and PRB-specific molecular mechanisms and as a database for subsequent experiments to identify novel PRA and PRB interacting proteins that differentially mediated different biological functions in breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Receptores de Progesterona/metabolismo , Aminoácidos/química , Linhagem Celular Tumoral , Feminino , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Receptores de Progesterona/química , Técnicas do Sistema de Duplo-Híbrido
13.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923443

RESUMO

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid-liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the ARPIN locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.


Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Tanquirases/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Tanquirases/química , Técnicas do Sistema de Duplo-Híbrido
14.
Plant Physiol ; 185(2): 369-384, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33721896

RESUMO

Jasmonates (JAs) are plant hormones that regulate the biosynthesis of many secondary metabolites, such as hydroxycinnamic acid amides (HCAAs), through jasmonic acid (JA)-responsive transcription factors (TFs). HCAAs are renowned for their role in plant defense against pathogens. The multidrug and toxic compound extrusion transporter DETOXIFICATION18 (DTX18) has been shown to mediate the extracellular accumulation of HCAAs p-coumaroylagmatine (CouAgm) at the plant surface for defense response. However, little is known about the regulatory mechanism of DTX18 gene expression by TFs. Yeast one-hybrid screening using the DTX18 promoter as bait isolated the key positive regulator redox-responsive TF 1 (RRTF1), which is a member of the AP2/ethylene-response factor family of proteins. RRTF1 is a JA-responsive factor that is required for the transcription of the DTX18 gene, and it thus promotes CouAgm secretion at the plant surface. As a result, overexpression of RRTF1 caused increased resistance against the fungus Botrytis cinerea, whereas rrtf1 mutant plants were more susceptible. Using yeast two-hybrid screening, we identified the BTB/POZ-MATH (BPM) protein BPM1 as an interacting partner of RRTF1. The BPM family of proteins acts as substrate adaptors of CUL3-based E3 ubiquitin ligases, and we found that only BPM1 and BPM3 were able to interact with RRTF1. In addition, we demonstrated that RRTF1 was subjected to degradation through the 26S proteasome pathway and that JA stabilized RRTF1. Knockout of BPM1 and BPM3 in bpm1/3 double mutants enhanced RRTF1 accumulation and DTX18 gene expression, thus increasing resistance to the fungus B. cinerea. Our results provide a better understanding of the fine-tuned regulation of JA-induced TFs in HCAA accumulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Botrytis/fisiologia , Ácidos Cumáricos/metabolismo , Doenças das Plantas/imunologia , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Amidas/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Mutação , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
16.
Methods Mol Biol ; 2297: 147-154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33656678

RESUMO

The LexA-based yeast two-hybrid system is one of the most powerful techniques used to detect blue light-dependent protein-protein interactions. In Arabidopsis, many protein-protein interactions in blue light signaling pathway were identified using this system. Here we present an easy and efficient method of the LexA-based yeast two-hybrid assay for testing protein-protein interactions in a blue light-dependent manner.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Cor , Vetores Genéticos , Leucina/metabolismo , Luz , Plasmídeos , Saccharomyces cerevisiae/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Transformação Genética , beta-Galactosidase/análise
17.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672238

RESUMO

Mutations in the Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in NKCC2 maturation and cell surface expression. Yet, the molecular mechanisms involved in this process remain obscure. Here, we report the identification of chaperone stress 70 protein (STCH) and the stress-inducible heat shock protein 70 (Hsp70), as two novel binding partners of the ER-resident form of NKCC2. STCH knock-down increased total NKCC2 expression whereas Hsp70 knock-down or its inhibition by YM-01 had the opposite effect. Accordingly, overexpressing of STCH and Hsp70 exerted opposite actions on total protein abundance of NKCC2 and its folding mutants. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, Hsp70 co-expression increased NKCC2 maturation. Interestingly, treatment by protein degradation inhibitors revealed that in addition to the proteasome, the ER associated degradation (ERAD) of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway. In summary, our data are consistent with STCH and Hsp70 having differential and antagonistic effects with regard to NKCC2 biogenesis. These findings may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Animais , Síndrome de Bartter/genética , Sítios de Ligação , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Humanos , Rim/citologia , Mutação , Gambás , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Membro 1 da Família 12 de Carreador de Soluto/genética , Técnicas do Sistema de Duplo-Híbrido
18.
Plant Sci ; 304: 110747, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568292

RESUMO

Anthocyanin provides a red color for apple and health benefit for human. To better understand the molecular mechanisms of regulating apple color formation, we analyzed 27 transcriptomes of fruit skin from three cultivars 'Huashuo' (red-skinned), 'Hongcuibao' (red-skinned), and 'Golden Delicious' (yellow-skinned) at 0, 2, and 6 days after bag removal. Using pairwise comparisons and weighted gene co-expression network analyses (WGCNA), we constructed 17 co-expression modules. Among them, a specific module was negatively correlated to anthocyanin accumulation. The genes in the module are enriched in flavonoid biosynthesis pathways. These pathway genes were used to construct gene co-expression network of anthocyanin accumulation. Finally, a R2R3-MYB repressor designated MdMYB28 was identified as a key hub gene in the anthocyanin metabolism network. During the anthocyanin accumulation of apple fruit skin reaching a peak, MdMYB28 expression level was negatively correlated with the anthocyanin content. MdMYB28 was shown to directly bind to the promoter of MdMYB10 in yeast one-hybrid analyses. Over-expression of MdMYB28 decreased the anthocyanin biosynthesis in tobacco flower petals, suggesting that MdMYB28 acts as a negatively regulator of anthocyanin biosynthesis.


Assuntos
Flavonoides/metabolismo , Frutas/metabolismo , Genes de Plantas/genética , Malus/genética , Epiderme Vegetal/metabolismo , Antocianinas/metabolismo , Regulação da Expressão Gênica de Plantas , Malus/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Tabaco , Transcriptoma , Técnicas do Sistema de Duplo-Híbrido
19.
Plant Sci ; 304: 110804, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568303

RESUMO

Adverse environmental conditions such as drought stress greatly limit the growth and production of crops worldwide. In this study, SlGRAS4, a drought stress-responsive GRAS gene from tomato (Solanum lycopersicum) was functionally characterized. Repressing SlGRAS4 (SlGRAS4-RNAi) increased sensitivity to drought stress, whereas overexpressing SlGRAS4 (SlGRAS4-OE) in tomato enhanced tolerance of this stress. Under stress condition SlGRAS4-OE plants accumulated much less ROS than wild-type and SlGRAS4-RNAi plants. Numerous dehydration induced ROS-scavenging genes were upregulated in SlGRAS4-OE plants after drought stress, implying that SlGRAS4 confers drought tolerance by modulating ROS homeostasis. On the other hand, there are several abscisic acid (ABA)-responsive elements in SlGRAS4 promoter, the relative expression of ABA signaling genes including SlPYLs, SlPP2Cs and SlSnRK2s were verified in WT and transgenic plants both under normal and drought stress, the changed drought sensitivity of transgenic plants was mainly caused by SlSnRK2s, the positive regulators of ABA signaling. Our results suggested that SlGRAS4 directly binds to and activates SlSnRK2.4 promoter, belongs to subclass III SnRK2s, which play crucial role in ABA signaling. Protein studies revealed that SlSnRK2.4 interacts with SlAREB1 and SlAREB2, the major downstream transcription factors of ABA-dependent signaling pathway. SlGRAS4 therefore confers drought tolerance may be through SnRK2-AREB pathway.


Assuntos
Ácido Abscísico/metabolismo , Genes de Plantas/fisiologia , Lycopersicon esculentum/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Transdução de Sinais , Desidratação , Germinação , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Transcriptoma , Técnicas do Sistema de Duplo-Híbrido
20.
Plant Sci ; 304: 110806, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568306

RESUMO

Control of gene transcription is crucial to regulate plant growth and development events, such as flowering, leaf senescence, and seed germination. Here we identified a NAC transcription factor (ScNAC23) isolated from sugarcane (cv. ROC22). Analysis by qRT-PCR indicated that ScNAC23 expression was strongly induced in mature leaves and flowering varieties and was also responsive to exogenous treatment with the hormone gibberellin (GA). Ectopic expression of ScNAC23 in Arabidopsis accelerated bolting, flowering, and leaf senescence compared to wild type plants. Furthermore, Arabidopsis overexpressed ScNAC23 were more sensitive to GA than the wild type, and exogenous GA significantly accelerated flowering and senescence in the ScNAC23-overexpressed ones. A direct interaction between ScNAC23 and ScGAI, an inhibitor of GA signaling, was confirmed by yeast-two hybrid, bimolecular fluorescence complementation, and GST-pull down assay. The putative GA-ScNAC23-LFY/SAGs regulator module might provide a new sight into the molecular action of GA to accelerating flowering and leaf senescence in sugarcane.


Assuntos
Flores/crescimento & desenvolvimento , Giberelinas/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/fisiologia , Saccharum/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Envelhecimento , Arabidopsis , Clorofila/metabolismo , Clonagem Molecular , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Saccharum/genética , Saccharum/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
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