المؤلفون: Mingsheng Qi, Haiyue Yu, Melissa Bredow, Aline Sartor Chicowski, Letícia Dias Fields, Steven A. Whitham

المصدر: Molecular Plant-Microbe Interactions, Vol 37, Iss 3, Pp 227-231 (2024)

مصطلحات موضوعية: effector–effector interactions, metaeffector, soybean rust, split-luciferase complementation, yeast-two-hybrid, Microbiology, QR1-502, Botany, QK1-989

الوصف: The multifaceted role of pathogen-encoded effectors in plant–pathogen interactions is complex and not fully understood. Effectors operate within intricate host environments, interacting with host proteins and other effectors to modulate virulence. The complex interplay between effectors raises the concept of metaeffectors, wherein some effectors regulate the activity of others. While previous research has demonstrated the importance of effector repertoires in pathogen virulence, only a limited number of studies have investigated the interactions between these effectors. This study explores the interactions among Phakopsora pachyrhizi effector candidates (PpECs). P. pachyrhizi haustorial transcriptome analysis identified a collection of predicted PpECs. Among these, PpEC23 was found to interact with PpEC48, prompting further exploration into their potential interaction with other effectors. Here, we utilized a yeast two-hybrid screen to explore protein–protein interactions between PpECs. A split-luciferase complementation assay also demonstrated that these interactions could occur within soybean cells. Interestingly, PpEC48 displayed the ability to interact with several small cysteine-rich proteins (SCRPs), suggesting its affinity for this specific class of effectors. We show that these interactions involve a histidine-rich domain within PpEC48, emphasizing the significance of structural motifs in mediating effector interactions. The unique nature of PpEC48, showing no sequence matches in other organisms, suggests its relatively recent evolution and potential orphan gene status. Our work reveals insights into the intricate network of interactions among P. pachyrhizi effector–effector interactions. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/0894-0282Test; https://doaj.org/toc/1943-7706Test

الوصول الحر: https://doaj.org/article/16c93cf1e3874c46acee6124f82fecaaTest

المؤلفون: Changhua Shang, Bingbing Pang, Jin Zhang, Lihong Yu, Shanling Gan, Yujia Li, Haifeng Wu

المصدر: Frontiers in Marine Science, Vol 11 (2024)

مصطلحات موضوعية: Dunaliella parva, AP2, yeast two-hybrid system, interacting proteins, carotenoid biosynthesis, Science, General. Including nature conservation, geographical distribution, QH1-199.5

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/articles/10.3389/fmars.2024.1448420/fullTest; https://doaj.org/toc/2296-7745Test

الوصول الحر: https://doaj.org/article/7c27b119b3ad4fb298adfb3a93d753fbTest

المؤلفون: Zhijun Weng, Xiaoyu Zheng, Yifan Liang, Xiongnan Chen, Qin Peng, Guihong Zhang, Lang Gong, Zezhong Zheng

المصدر: Frontiers in Microbiology, Vol 15 (2024)

مصطلحات موضوعية: African swine fever virus, p72, virus-host protein interaction, yeast two hybrid, coimmunoprecipitation, confocal localization, Microbiology, QR1-502

الوصف: IntroductionAfrican swine fever virus (ASFV) is a highly contagious virus that spreads rapidly and has a mortality rate of up to 100% in domestic pigs, leading to significant economic losses in the pig industry. The major capsid protein p72 of ASFV plays a critical role in viral invasion and immune evasion.MethodsIn this study, we used yeast two-hybrid screening to identify host proteins interacting with p72 in porcine alveolar macrophages (PAMs) and verified these proteins using confocal microscopy and immunoprecipitation techniques.Results and DiscussionWe validated 13 proteins that interact with p72, including CD63, B2M, YTHDF2, FTH1, SHFL, CDK5RAP3, VIM, PELO, TIMP2, PHYH, C1QC, CMAS, and ERCC1. Enrichment analysis and protein-protein interaction network analysis of these interacting proteins revealed their involvement in virus attachment, invasion, replication, assembly, and immune regulation. These findings provide new insights into the function of p72 and valuable information for future research on the interaction between ASFV and host proteins.

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/articles/10.3389/fmicb.2024.1370417/fullTest; https://doaj.org/toc/1664-302XTest

الوصول الحر: https://doaj.org/article/0d5abe76daea4db5973ce603c8dcac3fTest

المؤلفون: Shulan Huang, Hongning Zhang, Wen Chen, Jiawei Wang, Zhen Wu, Meiqi He, Jian Zhang, Xiang Hu, Shuanglin Xiang

المصدر: Current Issues in Molecular Biology, Vol 45, Iss 10, Pp 8215-8226 (2023)

مصطلحات موضوعية: Tnfaip1, zebrafish embryos, cDNA library, yeast two-hybrid system, interacting protein, Biology (General), QH301-705.5

الوصف: TNFAIP1 regulates cellular biological functions, including DNA replication, DNA repair, and cell cycle, by binding to target proteins. Identification of Tnfaip1-interacting proteins contributes to the understanding of the molecular regulatory mechanisms of their biological functions. In this study, 48 hpf, 72 hpf, and 96 hpf wild-type zebrafish embryo mRNAs were used to construct yeast cDNA library. The library titer was 1.12 × 107 CFU/mL, the recombination rate was 100%, and the average length of the inserted fragments was greater than 1000 bp. A total of 43 potential interacting proteins of Tnfaip1 were identified using zebrafish Tnfaip1 as a bait protein. Utilizing GO functional annotation and KEGG signaling pathway analysis, we found that these interacting proteins are mainly involved in translation, protein catabolic process, ribosome assembly, cytoskeleton formation, amino acid metabolism, and PPAR signaling pathway. Further yeast spotting analyses identified four interacting proteins of Tnfaip1, namely, Ubxn7, Tubb4b, Rpl10, and Ybx1. The Tnfaip1-interacting proteins, screened from zebrafish embryo cDNA in this study, increased our understanding of the network of Tnfaip1-interacting proteins during the earliest embryo development and provided a molecular foundation for the future exploration of tnfaip1’s biological functions.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1467-3045/45/10/518Test; https://doaj.org/toc/1467-3037Test; https://doaj.org/toc/1467-3045Test

الوصول الحر: https://doaj.org/article/8e59b30d485e4917805e6f2ee4f58f19Test

المؤلفون: Paloma Salinas, Sirine Bibak, Raquel Cantos, Lorena Tremiño, Carmen Jerez, Trinidad Mata-Balaguer, Asunción Contreras

المصدر: International Journal of Molecular Sciences, Vol 25, Iss 10, p 5429 (2024)

مصطلحات موضوعية: protein-protein interaction, nitrogen interaction network, Synechococcus elongatus PCC7942, yeast two-hybrid, bacterial two-hybrid, Biology (General), QH301-705.5, Chemistry, QD1-999

الوصف: Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a “false positive”, the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1422-0067/25/10/5429Test; https://doaj.org/toc/1661-6596Test; https://doaj.org/toc/1422-0067Test

الوصول الحر: https://doaj.org/article/b85a4327c1cc4cbd855b3fc7c9a7f8d3Test

المؤلفون: Nazife Gelmez, Elif Çağlayan, Kadir Turan

المصدر: Acta Virologica, Vol 67 (2024)

مصطلحات موضوعية: influenza A viruses, influenza RdRp, PA protein, β-actin, yeast two-hybrid, Biology (General), QH301-705.5

الوصف: Influenza A viruses are enveloped viruses with a genome of eight single-stranded negative-sense RNA molecules. In virions, RNA segments are found as vRNPs associated with NP proteins. The RdRp enzyme, which catalyzes the replication/transcription of the viral genome, is carried as attached to vRNPs. In this study, it was demonstrated that the PA subunit of the viral RdRp interacts with β-actin proteins by the yeast two-hybrid assay. It was shown that the amino-terminal domains of the β-actin protein bind to the carboxy-terminal moiety of the viral PA protein in the mammalian cells. The results were supported by in silico analysis. Over-expression of the β-actin protein was found to have a negative effect on the viral RdRp activity in mini-replicon, but its mechanism of action has remained unknown. The results suggest that the interaction of β-actin and PA protein, a component of vRNPs, may have a role in the intracellular trafficking of the influenza vRNPs and/or viral transcription.

وصف الملف: electronic resource

العلاقة: https://www.frontierspartnerships.org/articles/10.3389/av.2023.11890/fullTest; https://doaj.org/toc/1336-2305Test

الوصول الحر: https://doaj.org/article/e774da6b91a4400bba96231d30c9e265Test

المؤلفون: Raktim Ghosh, Pinaki Biswas, Abhinaba Chakraborty, Suchetana Pal, Moubonny Das, Somasri Dam

المصدر: Current Research in Biotechnology, Vol 7, Iss , Pp 100216- (2024)

مصطلحات موضوعية: Aurora kinase, Protein–protein interaction, Yeast two hybrid, Molecular docking, Entamoeba histolytica, Biotechnology, TP248.13-248.65

الوصف: Biomolecular interactions among proteins are fundamental for all cellular functions. The chromosome segregation proteins are the key regulators of inherent functions in the living cells. Aurora kinases have drawn much interest as possible drug targets in higher eukaryotes. The human pathogen, E. histolytica is the causative agent of amoebiasis, and a major health concern in developing countries. However, there is no vaccine against it and the popular drugs- metronidazole, tinidazole etc. show significant side effects in humans. To identify new controlling agents, we must have a thorough knowledge about the cell cycle regulatory proteins of E. histolytica, as many unusual cell cycle events can be found in this parasite, that do not happen in human cells. This study describes the first comprehensive analysis of the interaction between an aurora kinase protein and a BAR homology domain containing protein. Fes/CIP4 and EFC/F-BAR homology domain (FCH) containing protein, EhABP has been identified as a novel interactor of EhAK7, an aurora kinase homolog from E. histolytica by yeast two-hybrid screening against the cDNA library of E. histolytica and their interaction has been proved by in vitro binding assay. Both the N and C-terminus of EhAK7 are responsible for this interaction. We found the reduced expression of EhAK7 and EhABP genes, defects in actin filament organization and irregular-shaped nucleus in the trophozoites treated with an aurora kinase inhibitor VX-680. This indicates that EhAK7 play an important role in the cytokinesis of E. histolytica through the interaction with a BAR homology domain containing protein, EhABP.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S259026282400042XTest; https://doaj.org/toc/2590-2628Test

الوصول الحر: https://doaj.org/article/c938472d76364f719663a1924594b079Test

المؤلفون: Xuesong Li, Oliver Weth, Martin Haimann, Max F. Möscheid, Theresa S. Huber, Christoph G. Grevelding

المصدر: Microbiology Spectrum, Vol 12, Iss 1 (2024)

مصطلحات موضوعية: schistosomes, G protein-coupled receptor, neuropeptide, membrane-anchored ligand and receptor yeast two-hybrid system, neuronal signaling, Microbiology, QR1-502

الوصف: ABSTRACT Schistosomes are parasitic flatworms that cause schistosomiasis, a neglected tropical disease of worldwide importance. Since standard treatment of schistosomiasis relies on a single drug, praziquantel, alternative drugs are needed. G protein-coupled receptors (GPCRs) represent promising targets for new anthelmintics. Although GPCRs represent a prominent receptor class in schistosomes, functional studies are limited just as knowledge about their ligands. Candidate ligands are neuropeptides acting as neurotransmitters, neuromodulators, or hormones in the nervous system. Transcriptomics studies in Schistosoma mansoni indicated that nearly all neuropeptide genes (Sm_npps) and a subgroup of GPCRs exhibited a sex- and pairing-dependent expression profile. Among these was the rhodopsin orphan GPCR20 (SmGPCR20), which we characterized in our study. Using a yeast two-hybrid-based approach, we identified specific interactions between SmGPCR20 and two neuropeptides SmNPP26 and SmNPP40. As analyzed by qRT-PCR, Smgpcr20, Smnpp26, and Smnpp40 showed sex- and/or pairing-influenced expression. Whole-mount in situ hybridization exhibited transcripts of these genes in neuronal cells, subtegumental area, and parenchyma of both sexes. Furthermore, we received indication for co-localization of transcripts of these genes in the anterior “head” region of single-sex females and in particular patterns along the worm body indicating neuronal expression. RNA interference (RNAi) with combinations of double-stranded RNAs against the three genes resulted in reduced egg production. Confocal microscopy revealed morphologic changes in the female gonads. Furthermore, RNAi in first-time paired females caused a reduced length of females after double knockdown of SmGPCR20 and SmNPP26 and changes in the ovary. In addition, we found reduced transcript levels of egg formation-associated and gonad-specifically transcribed genes and the stem-cell marker nanos-1. The obtained results suggest that SmNPP26 and SmNPP40 are potential ligands of SmGPCR20 and that this GPCR in combination with both neuropeptides affects egg production, oogenesis, and growth of S. mansoni females. IMPORTANCE Schistosomes cause schistosomiasis, one of the neglected tropical diseases as defined by the WHO. For decades, the treatment of schistosomiasis relies on a single drug, praziquantel. Due to its wide use, there is justified fear of resistance against this drug, and a vaccine is not available. Besides its biological relevance in signal transduction processes, the class of G protein-coupled receptors (GPCRs) is also well suited for drug design. Against this background, we characterized one GPCR of Schistosoma mansoni, SmGPCR20, at the molecular and functional level. We identified two potential neuropeptides (NPPs) as ligands, SmNPP26 and SmNPP40, and unraveled their roles, in combination with SmGPCR20, in neuronal processes controlling egg production, oogenesis, and growth of S. mansoni females. Since eggs are closely associated with the pathogenesis of schistosomiasis, our results contribute to the understanding of processes leading to egg production in schistosomes, which is under the control of pairing in this exceptional parasite.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2165-0497Test

الوصول الحر: https://doaj.org/article/e3ef3602c3494102b4fac54d05c887acTest

المؤلفون: Salinas, Paloma, Bibak, Sirine, Cantos, Raquel, Tremiño, Lorena, Jerez García, Carmen, Mata Balaguer, Trinidad, Contreras, Asunción

المساهمون: Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Transducción de Señales en Bacterias

مصطلحات موضوعية: Protein-protein interaction, Nitrogen interaction network, Synechococcus elongatus PCC7942, Yeast two-hybrid, Bacterial two-hybrid

الوصف: Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a “false positive”, the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system. ; This work was supported by the grant PID2020-118816GB-I00, funded by MCIN/AEI/10.13039/501100011033 from the Spanish Government, grants VIGROB23-126 and GRE20-04-C from the University of Alicante to A.C. C.J. was the recipient of a PhD fellowship (ACIF/2019/045) from Conselleria d’Innovació, Universitats, Ciència i Societat Digital of the Generalitat Valenciana. S.B. was supported by a National Grant from the Algerian Ministry of Higher Education and Scientific Research.

العلاقة: https://doi.org/10.3390/ijms25105429Test; info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-118816GB-I00; International Journal of Molecular Sciences. 2024, 25(10): 5429. https://doi.org/10.3390/ijms25105429Test; http://hdl.handle.net/10045/143363Test

الإتاحة: https://doi.org/10.3390/ijms25105429Test
http://hdl.handle.net/10045/143363Test

المؤلفون: Qijun Wu, Yingying Lei, Ya Zuo, Ji Zhang, Fenglin Guo, Weize Xu, Tanghui Xie, Dang Wang, Guiqing Peng, Xiangru Wang, Huanchun Chen, Zhenfang Fu, Gang Cao, Jinxia Dai

المصدر: mSystems, Vol 8, Iss 6 (2023)

مصطلحات موضوعية: African swine fever virus, host immune pathway, protein-protein interaction, high-throughput yeast two-hybrid screening, Microbiology, QR1-502

الوصف: ABSTRACTAfrican swine fever virus (ASFV) causes severe acute hemorrhagic disease with high mortality in domestic pigs but minimal or no symptoms to warthogs, for which the underlying mechanism remains elusive. ASFV encodes numerous proteins to disturb host immune responses via interacting with host proteins. In this study, we deciphered the comprehensive protein-protein interaction (PPI) network between ASFV and host immune pathways by the recombination-based library vs library high-throughput yeast two-hybrid screening. This PPI network contains both ASFV-host PPI and ASFV-ASFV PPI information, providing a comprehensive ASFV-host interactome landscape. We further explored the ASFV-host PPI difference between domestic pigs and warthogs. Moreover, the inhibitory effect of ASFV proteins in the PPI with cGAS-STING pathway on IFN-I and NF-κB promoter activity was screened to investigate the ASFV-host PPI functions. Our work will help the exploration of ASFV pathogenesis and the development of anti-ASFV vaccine and ASFV-resistant transgenic pigs.IMPORTANCEAfrican swine fever (ASF), caused by African swine fever virus (ASFV), has become a major crisis for the pork industry in recent years. The mechanism for ASFV pathology and the clinical symptoms difference of ASF between domestic pigs and reservoir hosts remain to be elucidated. We deciphered the comprehensive protein-protein interaction (PPI) network between ASFV and host immune pathways. The intensive PPI network contained both ASFV-host immune pathway PPI and ASFV-ASFV PPI information, providing a comprehensive ASFV-host interaction landscape. Furthermore, the ASFV-host PPI difference between domestic pigs and warthogs was explored, which will be instructive for exploring essential candidates involved in ASFV pathology. Moreover, we screened the inhibitory effect of ASFV proteins in the PPI with cGAS-STING pathway on IFN-I and NF-κB, further providing possible functions of ASFV-host PPI network in innate immune regulation.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2379-5077Test

الوصول الحر: https://doaj.org/article/0226ec3b95984096a32c05dd7013185aTest