Two-hybrid Research Articles

Ubiquitin Ligase U-Box51 Positively Regulates Drought Stress in Potato (Solanum tuberosum L.)

The ubiquitin-proteasome system (UPS) is a key protein degradation pathway in eukaryotes, in which E3 ubiquitin ligases mediate protein ubiquitination, directly or indirectly targeting substrate proteins to regulate various biological processes, including plant growth, hormone signaling, immune responses, and adaptation to abiotic stress. In this study, we identified plant U-box protein 51 in Solanum tuberosum (StPUB51) as an E3 ubiquitin ligase through transcriptomic analysis, and used it as a candidate gene for gene-function analysis. Quantitative real-time PCR (qRT-PCR) was used to examine StPUB51 expression across different tissues, and its expression patterns under simulated drought stress induced by polyethylene glycol (PEG 6000) were assessed. Transgenic plants overexpressing StPUB51 and plants with down-regulated StPUB51 expression were generated to evaluate drought tolerance. The activities of key antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) as well as malondialdehyde (MDA) content in transgenic plants’ leaves were measured under drought conditions. Protein–protein interactions involving StPUB51 were explored via yeast two-hybrid (Y2H) screening, with interaction verification by bimolecular fluorescence complementation (BiFC). StPUB51 was predominantly expressed in stems, with lower expression observed in tubers, and its expression was significantly upregulated in response to 20% PEG-6000 simulated drought. Subcellular localization assays revealed nuclear localization of the StPUB51 protein. Under drought stress, StPUB51-overexpressing plants exhibited enhanced SOD, POD, and CAT activities and reduced MDA levels, in contrast to plants with suppressed StPUB51 expression. Y2H and BiFC analyses identified two interacting proteins, StSKP2A and StGATA1, which may be functionally linked to StPUB51. Collectively, these findings suggest that StPUB51 plays a positive regulatory role in drought tolerance, enhancing resilience in potato growth and stress adaptation.

Host protein PRPS2 interact with the non-structural protein p17 of Avian Reovirus and promote viral replication

Avian reovirus (ARV) is highly prevalent in healthy poultry flocks and has been linked to viral arthritis/tendonitis, dwarf syndrome, chronic respiratory disease, and immunosuppression in avian species, resulting in significant economic losses within the poultry industry. The non-structural protein p17 encoded by ARV induces cellular autophagy and facilitates viral proliferation, playing a pivotal role in viral pathogenesis. To further elucidate the pathogenic mechanism basis of ARV p17 protein function, we employed a yeast two-hybrid system to identify Phosphoribosyl pyrophosphate synthetase 2 (PRPS2) as an interacting host protein with p17. In this study, we validated the interaction between PRPS2 and p17 using laser confocal microscopy, coimmunoprecipitation, and GST-Pulldown assays. Moreover, our findings demonstrate that the C-terminal region of PRPS2 is responsible for its binding to the p17 protein. Intriguingly, ARV infection significantly upregulated PRPS2 expression levels. Additionally, PRPS2 was shown to have a substantial impact on ARV replication; overexpression of PRPS2 increased ARV replication while knockdown of PRPS2 resulted in decreased quantities of ARV particles. Furthermore, our findings suggest that this process involves cellular apoptosis as a potential mechanism underlying these observations. Overall, this research provides valuable insights into elucidating the function of the p17 protein and sheds light on the pathogenic mechanism involving ARV-induced cellular apoptosis while offering novel perspectives for exploring therapeutic targets against ARV.

The receptor MIK2 interacts with the kinase RKS1 to control quantitative disease resistance to Xanthomonas campestris.

Molecular mechanisms underlying qualitative resistance have been intensively studied. In contrast, although quantitative disease resistance (QDR) is a common, durable and broad-spectrum form of immune responses in plants, only a few related functional analyses have been reported. The atypical kinase Resistance related KinaSe1 (RKS1) is a major regulator of QDR to the bacterial pathogen Xanthomonas campestris (Xcc) and is positioned in a robust protein-protein decentralized network in Arabidopsis (Arabidopsis thaliana). Among the putative interactors of RKS1 found by yeast two-hybrid screening, we identified the receptor-like kinase MDIS1-Interacting Receptor-like Kinase 2 (MIK2). Here, using multiple complementary strategies including protein-protein interaction tests, mutant analysis, and network reconstruction, we report that MIK2 is a component of RKS1--mediated QDR to Xcc. First, by co-localization experiments, co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC), we validated the physical interaction between RKS1 and MIK2 at the plasma membrane. Using mik2 mutants, we showed that MIK2 is required for QDR and contributes to resistance to the same level as RKS1. Interestingly, a catalytic mutant of MIK2 interacted with RKS1 but was unable to fully complement the mik2-1 mutant phenotype in response to Xcc. Finally, we investigated the potential role of the MIK2-RKS1 complex as a scaffolding component for the coordination of perception events by constructing a RKS1-MIK2 centered protein-protein interaction network. Eight mutants corresponding to seven RKs in this network showed a strong alteration in QDR to Xcc. Our findings provide insights into the molecular mechanisms underlying the perception events involved in QDR to Xcc.

The wheat CC-NBS-LRR protein TaRGA3 confers resistance to stripe rust by suppressing ascorbate peroxidase 6 activity

Abstract Nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune receptors that activate innate immune responses upon sensing pathogen attack. However, the molecular mechanisms by which NLR proteins initiate downstream signal transduction pathways to counteract pathogen invasion remain poorly understood. In this study, we identified the wheat (Triticum aestivum) NLR protein Resistance Gene Analogs3 (TaRGA3), which was significantly upregulated during Puccinia striiformis f. sp. tritici (Pst) infection. TaRGA3 and its coiled-coil (CC) domain, localized to the cytoplasm and nucleus, can induce cell death in Nicotiana benthamiana. Virus-induced gene silencing and overexpression suggested that TaRGA3 contributed to wheat resistance to stripe rust by facilitating reactive oxygen species (ROS) accumulation. Yeast 2-hybrid, luciferase complementation imaging, and co-immunoprecipitation assays revealed that TaRGA3 interacted with wheat protein Ascorbate Peroxidase 6 (TaAPX6). Further analysis showed that TaAPX6 specifically targeted the CC domain of TaRGA3. The TaRGA3–TaAPX6 interplay led to reduced enzyme activity of TaAPX6. Notably, TaAPX6 negatively regulated wheat resistance to Pst by removing excessive ROS accompanying Pst-induced hypersensitive responses. Our findings reveal that TaRGA3 responding to Pst infection confers enhanced wheat resistance to stripe rust, possibly by suppressing TaAPX6-modulated ROS scavenging, and demonstrate that TaRGA3 can be used to engineer stripe rust resistance in wheat.

A dispensable SepIVA orthologue in Streptomyces venezuelae is associated with polar growth and not cell division

BackgroundSepIVA has been reported to be an essential septation factor in Mycolicibacterium smegmatis and Mycobacterium tuberculosis. It is a coiled-coil protein with similarity to DivIVA, a protein necessary for polar growth in members of the phylum Actinomycetota. Orthologues of SepIVA are broadly distributed among actinomycetes, including in Streptomyces spp.ResultsTo clarify the role of SepIVA and its potential involvement in cell division in streptomycetes, we generated sepIVA deletion mutants in Streptomyces venezuelae and found that sepIVA is dispensable for growth, cell division and sporulation. Further, mNeonGreen-SepIVA fusion protein did not localize at division septa, and we found no evidence of involvement of SepIVA in cell division. Instead, mNeonGreen-SepIVA was accumulated at the tips of growing vegetative hyphae in ways reminiscent of the apical localization of polarisome components like DivIVA. Bacterial two-hybrid system analyses revealed an interaction between SepIVA and DivIVA. The results indicate that SepIVA is associated with polar growth. However, no phenotypic effects of sepIVA deletion could be detected, and no evidence was observed of redundancy with the other DivIVA-like coiled-coil proteins Scy and FilP that are also associated with apical growth in streptomycetes.ConclusionsWe conclude that S. venezuelae SepIVA, in contrast to the situation in mycobacteria, is dispensable for growth and viability. The results suggest that it is associated with polar growth rather than septum formation.

Transcription Factor Deformed Wings Is an Atg8a-Interacting Protein That Regulates Autophagy.

LC3 (microtubule-associated protein 1 light chain 3, called Atg8 in yeast and Drosophila) is one of the most well-studied autophagy-related proteins. LC3 controls the selectivity of autophagic degradation by interacting with LIR (LC3-interacting region) motifs also known as AIM (Atg8-interacting motifs) on selective autophagy receptors that carry cargo for degradation. Although the function of Atg8 family proteins is primarily cytoplasmic, they are also enriched in the nucleus. Despite the accumulating evidence indicating the presence of Atg8 proteins in the nucleus, the mechanisms by which they are targeted to the nucleus, their interactions with nuclear components, and their nuclear role in remain poorly understood. Here, we used yeast two-hybrid screening, and we identified transcription factor Deformed wings (Dwg) as an Atg8a-interacting protein in Drosophila. Dwg-Atg8a interaction is LIR motif-dependent. We have created Dwg Y129A/I132A LIR mutant flies and shown that they exhibit elevated autophagy, improved resistance to oxidative stress, and starvation. Our results provide novel insights into the transcriptional regulation of autophagy in Drosophila.

Identification of a novel locus qGW12/OsPUB23 regulating grain shape and weight in rice (Oryza sativa L.).

Key message A major quantitative trait locus (qGW12) for grain shape and weight has been isolated in rice, corresponding to LOC_Os12g17900/OsPUB23, and its encoded protein interacts with OsMADS1. Grain shape in rice is an important trait that influences both yield and quality. The primary determinants of grain shape are quantitative trait loci (QTLs) inherited from natural variation in crops. In recent years, much attention has been paid to the molecular role of QTLs in regulating grain shape and weight. In this study, we report the cloning and characterization of qGW12, a major QTL regulating grain shape and weight in rice, using a series of chromosome fragment substitution lines (CSSLs) derived from Oryza sativa indica cultivar 9311 (acceptor) and Oryza rufipogon Griff (donor). One CSSL line, Q187, harboring the introgression of qGW12, exhibited a significant decrease in grain-shape-related traits (including grain length and width) and thousand-grain weight compared to the cultivar 9311. Subsequent backcrossing of Q187 with 9311 resulted in the generation of secondary segregating populations, which were used to fine-map qGW12 to a 24-kb region between markers Seq-44 and Seq-48. Our data indicated that qGW12 encodes a previously unreported U-box type E3 ubiquitin ligase, designated OsPUB23, which exhibited E3 ubiquitin ligase activity. Overexpression of OsPUB23 in rice resulted in higher plant yield than the wild type due to an increase in grain size and weight. Conversely, loss of OsPUB23 function resulted in the opposite tendency. Yeast two-hybrid screening and split luciferase complementation assays revealed that OsPUB23 interacts with OsMADS1. The functional characterization of OsPUB23 provides new genetic resources for improving of grain yield and quality in crops.

HCV infection activates the proteasome via PA28γ acetylation and heptamerization to facilitate the degradation of RNF2, a catalytic component of polycomb repressive complex 1.

We previously reported that hepatitis C virus (HCV) infection or HCV core protein expression induces HOX gene expression by impairing histone H2A monoubiquitination via a proteasome-dependent reduction in the level of RNF2, a key catalytic component of polycomb repressive complex 1 (H. Kasai, K. Mochizuki, T. Tanaka, A. Yamashita, et al., J Virol 95:e01784-20, 2021, https://doi.org/10.1128/jvi.01784-20). In this study, we aimed to investigate the mechanism by which HCV infection accelerates RNF2 degradation. Yeast two-hybrid screening and an immunoprecipitation assay revealed that RNF2 is a PA28γ-binding protein. The proteasome activator PA28γ destabilized the RNF2 protein in a proteasome-dependent manner, since RNF2 degradation was impaired by PA28γ knockout or MG132 treatment. HCV infection or core protein expression reduced the levels of RNF2 and histone H2A K119 monoubiquitination and induced the expression of HOX genes in the presence of PA28γ, while PA28γ knockout reversed these changes. Treatment with a lysine acetyltransferase inhibitor inhibited the acetylation of PA28γ at K195 and the degradation of the RNF2 protein, while treatment with a lysine deacetylase inhibitor accelerated these events in a PA28γ-dependent manner. RNF2 protein degradation was increased by expression of the acetylation mimetic PA28γ mutant but not by expression of the acetylation-defective mutant or the proteasome activation-defective mutant. Furthermore, HCV infection or core protein expression facilitated the interaction between PA28γ and the lysine acetyltransferase CBP/p300 and then accelerated PA28γ acetylation and heptazmerization to promote RNF2 degradation. These data suggest that HCV infection accelerates the acetylation-dependent heptamerization of PA28γ to increase the proteasomal targeting of RNF2.IMPORTANCEHCV is a causative agent of HCV-related liver diseases, including hepatic steatosis, cirrhosis, and hepatocellular carcinoma. PA28γ, which, in heptameric form, activates the 20S core proteasome for the degradation of PA28γ-binding proteins, is responsible for HCV-related liver diseases. HCV core protein expression or HCV infection accelerates RNF2 degradation, leading to the induction of HOX gene expression via a decrease in the level of H2Aub on HOX gene promoters. However, the mechanism of RNF2 degradation in HCV-infected cells has not been clarified. The data presented in this study suggest that PA28γ acetylation and heptamerization are promoted by HCV infection or by core protein expression to activate the proteasome for the degradation of RNF2 and are responsible for HCV propagation. This study provides novel insights valuable for the development of therapies targeting both HCV propagation and HCV-related diseases.

Dual Assay Validation of Rosmarinus officinalis Extract as an Inhibitor of SARS-CoV-2 Spike Protein: Combining Pseudovirus Testing, Yeast Two-Hybrid, and UPLC-Q Exactive Orbitrap-MS Profiling.

This study evaluates the effectiveness of Traditional Chinese Medicine (TCM) extracts in blocking the interaction between the SARS-CoV-2 Spike protein and human ACE2 receptor, utilizing a dual-method approach to explore the antiviral potential of natural compounds. This work aims to evaluate the capability of TCM extracts in inhibiting the SARS-CoV-2 Spike protein and ACE2 receptor interaction using advanced biochemical assays. A dual-method screening approach was utilized, beginning with a pseudovirus assay to assess the inhibition capabilities of TCM extracts invitro, followed by a split-ubiquitin yeast two-hybrid (Y2H) system to validate interactions in live cells. Active compounds were characterized and quantified using UPLC-Q-Exactive-Orbitrap-MS. Among the 91 TCM extracts tested, Rosmarinus officinalis exhibited the most potent inhibition in both pseudovirus and Y2H assays, significantly reducing viral entry and disrupting the Spike-ACE2 interaction. Comprehensive chemical profiling via UPLC-Q-Exactive-Orbitrap-MS identified 132 compounds, including phenolics, flavonoids, and terpenoids. This research validates the use of TCM extracts in viral inhibition strategies, demonstrating the utility of integrating traditional remedies with modern scientific approaches to discover new therapeutic agents.

Strain-Specific Infection of Phage AP1 to Rice Bacterial Brown Stripe Pathogen Acidovorax oryzae.

Bacteriophage (phage) AP1 has been reported to effectively lyse Acidovorax oryzae, the causative agent of bacterial brown stripe in rice. However, phage AP1 exhibits strain-specific lysis patterns. In order to enhance the potential of phages for biological control of rice bacterial brown stripe, this study investigated the possible mechanism of strain-specific infection by characterizing phage AP1 and its susceptible (RS-2) and resistant (RS-1) strains. Based on the current classification standards and available database information, phage AP1 was classified into the class Caudoviricetes, and it is a kind of podophage. Comparative analysis of the susceptible and resistant strains showed no significant differences in growth kinetics, motility, biofilm formation, or effector Hcp production. Interestingly, the resistant strain demonstrated enhanced virulence compared to the susceptible strain. Prokaryotic expression studies indicated that six putative structural proteins of phage AP1 exhibited varying degrees of binding affinity (1.90-9.15%) to lipopolysaccharide (LPS). However, pull-down assays and bacterial two-hybrid analyses revealed that only gp66 can interact with four host proteins, which were identified as glycosyltransferase, RcnB, ClpB, and ImpB through immunoprecipitation and mass spectrometry analyses. The role of LPS in the specific infection mechanism of phage AP1 was further elucidated through the construction of knockout mutant strains and complementary strains targeting a unique gene cluster (wbzB, wbzC, wbzE, and wbzF) involved in LPS precursor biosynthesis. These findings provide novel insights into the mechanisms of phage-host specificity, which are crucial for the effective application of phage AP1 in controlling rice bacterial brown stripe.

MecA in Streptococcus mutans is a multi-functional protein.

MecA is known as an adaptor protein that works in concerto with ATPase ClpC and protease ClpP in the regulated proteolysis machinery. The results presented here provide further evidence that MecA in S. mutans, a keystone cariogenic bacterium, plays a significant role in its ability to facilitate mixed-species biofilm formation, a trait critical to its cariogenicity. Proteomics analysis, along with affinity pull-down and bacterial two-hybrid system, further confirm that MecA can also regulate S. mutans physiology and biofilm formation through pathways independent of the Clp proteolytic machinery, although how it functions independently of Clp awaits further investigation.

The homeostasis of AtMYB4 is maintained by ARA4, HY5, and CAM7 during Arabidopsis seedling development.

Calmodulin7 (CAM7) is a key transcription factor of Arabidopsis seedling development. CAM7 works together with HY5 bZIP protein to promote photomorphogenesis at various wavelengths of light. In this study, we show that AtMYB4, identified from a yeast two-hybrid screen, physically interacts with CAM7 and works as a positive regulator of photomorphogenesis at various wavelengths of light. CAM7 and HY5 directly bind to the promoter of AtMYB4 to promote its expression for photomorphogenic growth. On the other hand, ARA4, identified from the same yeast two-hybrid screen, works as a negative regulator of photomorphogenic growth specifically in white light. The double mutant analysis reveals that the altered hypocotyl elongation of atmyb4 and ara4 is either partly or completely suppressed by additional loss of function of CAM7. Furthermore, ARA4 genetically interacts with AtMYB4 in an antagonistic manner to suppress the elongated hypocotyl phenotype of atmyb4. The transactivation studies reveal that while CAM7 activates the promoter of AtMYB4 in association with HY5, ARA4 negatively regulates AtMYB4 expression. Taken together, these results demonstrate that working as a negative regulator of photomorphogenesis, ARA4 plays a balancing act on CAM7 and HY5-mediated regulation of AtMYB4.

A comprehensive two-hybrid analysis to explore the Legionella pneumophila effector-effector interactome.

Secreted bacterial effector proteins are typically viewed as modulators of host activity, entering the host cytosol to physically interact with and modify the activity of one or more host proteins in support of infection. A growing body of evidence suggests that a subset of effectors primarily function to modify the activities of other effectors inside the host. These "effectors of effectors" or metaeffectors are often identified through experimental serendipity during the study of canonical effector function against the host. We previously performed the first global effector-wide genetic interaction screen for metaeffectors within the arsenal of Legionella pneumophila, an intracellular bacterial pathogen with over 300 effectors. Here, using a high-throughput, scalable methodology, we present the first global interaction network of physical interactions between L. pneumophila effectors. This data set serves as a complementary resource to identify and understand both the scope and nature of non-canonical effector activity within this important human pathogen.

The uncharacterized protein ZNF200 interacts with PRMT3 and aids its stability and nuclear translocation.

Protein arginine methyltransferase 3 (PRMT3), a type I arginine methyltransferase is localized predominantly in the cytoplasm and regulates different cellular functions. Nevertheless, PRMT3 also exhibits regulatory functions in the nucleus by interacting with the liver X receptor alpha (LXRα) and catalyzes asymmetric dimethylation modifications at arginine 3 of histone 4 (H4R3me2a). However, very little is known about the regulation of the versatile global regulator PRMT3 and how PRMT3 is translocated to the nucleus. In this study, we identified ZNF200, a hitherto uncharacterized protein, as a potential binding partner of PRMT3 through yeast two-hybrid screening. We confirmed the interaction of PRMT3 with ZNF200 using immunoprecipitation and in vitro pull-down experiments. GST pull-down experiments and molecular docking studies revealed that the N-terminal zinc finger domain of PRMT3 binds to the C-terminal zinc finger regions of ZNF200. Furthermore, the evolutionary conservation of the Znf domain of PRMT3 correlates with the emergence of ZNF200 in mammals. We found that ZNF200 stabilizes PRMT3 by inhibiting its proteasomal degradation. ZNF200, a nuclear-predominant protein, promotes the nuclear translocation of PRMT3, leading to the global increase of H4R3me2a modifications. These findings imply that ZNF200 is a critical regulator of the steady-state levels and nuclear and epigenetic functions of PRMT3.

Extracellular matrix protein 1 binds to connective tissue growth factor against liver fibrosis and ductular reaction.

Extracellular matrix protein 1 (ECM1) can inhibit TGFβ activation, but its antifibrotic action remains largely unknown. This study aims to investigate ECM1 function and its physical interaction with the profibrotic connective tissue growth factor (CTGF) in fibrosis and ductular reaction (DR). Ecm1 knockouts or animals that ectopically expressed this gene were subjected to induction of liver fibrosis and DR by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or α-naphthyl-isothiocyanate (ANIT). ECM1 and CTGF were also examined in the livers of patients with alcohol-associated liver disease (ALD) or ethanol-exposed animals that were fed the western diet for 4 months in the WDA model with liver pathology resembing ALD in patients. ECM1 bound to CTGF in yeast two-hybrid systems, cultured liver cells, and cholestatic livers damaged by DDC or α-naphthyl-isothiocyanate. This interaction blocked integrin αvβ6-mediated TGFβ activation, thereby reducing fibrotic responses in vitro. ECM1 downregulation was associated with biliary CTGF induction during human ALD progression. In experimental models, Ecm1 loss enhanced susceptibility to DDC-induced cholestasis with upregulation of Ctgf, αvβ6, alpha-smooth muscle actin, procollagen type I, serum transaminase, and total bilirubin levels in germline knockouts, whereas forced expression of this gene significantly attenuated DR and biliary fibrosis after the feeding of DDC or α-naphthyl-isothiocyanate containing diets. Moreover, ectopic Ecm1 inhibited not only alcohol-associated fibrosis but also TGFβ-mediated deregulation of hepatocyte nuclear factor 4α, preventing the production of the fetal p2 promoter-driven isoforms in the WDA model. We uncover a novel antifibrotic action by ECM1 that binds CTGF and inhibits integrin αvβ6-mediated TGFβ activation. Targeting its loss has therapeutic potential for the treatment of DR and liver fibrosis in chronic conditions, such as cholangiopathy and ALD.

The importin FocKap119 is required for fungal growth and pathogenicity of the Fusarium oxysporum f. sp. cubense via interaction with FocCks1

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of banana wilt disease, is one of the most devastating pathogens of bananas (Musa spp.). The nucleocytoplasmic trafficking of proteins and RNAs, mediated by karyopherins (Kaps), is essential for eukaryotes. However, little is known about how Kaps regulate fungal growth and pathogenicity. Here, a Kap119 homolog (FocKap119) was identified in the Foc tropical race 4 strain Foc302. Deletion of FocKap119 led to impaired vegetative growth, conidiation, cell wall integrity, and pathogenicity. Compared to the wild type, the ΔFocKap119 mutant showed increased sensitivity to oxidative stress but greater tolerant to osmotic stress. FocKap119 was found to interact with FocCks1 (cell cycle-dependent kinase subunit 1) in a yeast two-hybrid system, and deletion of FocCks1 showed similar phenotypes as ΔFocKap119. Moreover, whole-transcriptome analysis and qRT-PCR results indicated that deletion of FocKap119 resulted in down-regulation of genes related to key virulence factor biosynthesis, including fusaric acid and beauvericin biosynthetic gene clusters. The ΔFocKap119 mutant also triggered stronger plant defense responses during the early infection compared to the wild type. This study provides insights into how FocKap119 regulated growth, abiotic stress response, and pathogenicity in Foc.

Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast.

The yeast one-hybrid system (Y1H) is used extensively to identify DNA-protein interactions. The generation of large collections of open reading frames (ORFs) to be used as prey in screenings is not a bottleneck nowadays and can be carried out in-house or offered as a service by companies. However, the straightforward use of full gene promoters as baits to identify interacting proteins undermines the accuracy and sensitivity of the assay, especially in the case of multicellular eukaryotes. Therefore, it is paramount to implement procedures for efficient identification of suitable promoter fragments compatible with the Y1H assay. Here, we describe a workflow to identify biologically relevant conserved promoter fragments of Arabidopsis thaliana through simple and robust phylogenetic analyses. Additionally, we describe a manual method and its automated robotized version for rapid and efficient high-throughput Y1H screenings of arrayed ORF libraries with the identified DNA fragments. Moreover, this method can be scaled up or down and used for yeast two-hybrid screenings to search for possible interactors of proteins identified by the Y1H approach or any other protein of interest, altogether underscoring its suitability to build gene regulatory networks. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selection of DNA baits for Y1H screenings Basic Protocol 2: Y1H screenings with arrayed gene libraries Alternate Protocol: Automated screening with a liquid-handling robot.

Brucella secretory protein VceA promotes FOXO1 entry into the nucleus to shift host cell metabolism toward glycolysis.

Increased glycolytic metabolism is a key step in the reproduction of Brucella and the induction of brucellosis, however, little is known about how this process is regulated during infection. Forkhead box protein O1 (FOXO1) is a transcription factor that regulates energy metabolism. In this study, we employ the yeast two-hybrid system (Y2H) and immunoprecipitation (Co-IP) to reverse screen for the FOXO1 for the first time and identify interactions between FOXO1 and the Brucella secretory protein VceA. Our findings reveal that the Brucella secretory protein VceA colocalizes with FOXO1 in the cytoplasm. Additionally, we observe that infection of macrophages with Brucella abortus 2308 ( S2308) promotes FOXO1 entry into the nucleus, leading to a significant upregulation of glycolysis level in macrophage. Conversely, in a VceA mutant strain (S2308-ΔVceA), we note a significant reduction in the ability of FOXO1 to enter the nucleus, accompanied by a decrease in glycolysis level. Furthermore, Brucella interacts with FOXO1 through the secreted protein VceA, promoting the entry of FOXO1 into the nucleus and thereby altering host metabolic patterns. This study provides insights into the mechanisms by which Brucella invades host macrophages and induces unique metabolic changes. These insights may offer a novel rationale for developing metabolic therapeutic strategies for the treatment and prevention of related diseases.

A non-catalytic role of IPMK is required for PLCγ1 activation in T cell receptor signaling by stabilizing the PLCγ1-Sam68 complex

BackgroundPhospholipase C gamma 1 (PLCγ1) is an important mediator of the T cell receptor (TCR) and growth factor signaling. PLCγ1 is activated by Src family kinases (SFKs) and produces inositol 1,4,5-triphosphate (InsP3) from phosphatidylinositol 4,5-bisphosphate (PIP2). Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme with broad substrate specificity and non-catalytic activities that mediate various functional protein-protein interactions. Therefore, IPMK plays critical functions in key biological events such as cell growth. However, the contribution of IPMK to the activation of PLCγ1 in TCR signaling remains mostly unelucidated. The current study aimed to elucidate the functions of IPMK in TCR signaling and to uncover the mode of IPMK-mediated signaling action in PLCγ1 activation.MethodsConcanavalin A (ConA)-induced acute hepatitis model was established in CD4+ T cell-specific IPMK knockout mice (IPMKΔCD4). Histological analysis was performed to assess hepatic injury. Primary cultures of naïve CD4+ T cells were used to uncover the role of mechanisms of IPMK in vitro. Western blot analysis, quantitative real-time PCR, and flow cytometry were performed to analyze the TCR-stimulation-induced PLCγ1 activation and the downstream signaling pathway in naïve CD4+ T cells. Yeast two-hybrid screening and co-immunoprecipitation were conducted to identify the IPMK-binding proteins and protein complexes.ResultsIPMKΔCD4 mice showed alleviated ConA-induced acute hepatitis. CD4+ helper T cells in these mice showed reduced PLCγ1 Y783 phosphorylation, which subsequently dampens calcium signaling and IL-2 production. IPMK was found to contribute to PLCγ1 activation via the direct binding of IPMK to Src-associated substrate during mitosis of 68 kDa (Sam68). Mechanistically, IPMK stabilizes the interaction between Sam68 and to PLCγ1, thereby promoting PLCγ1 phosphorylation. Interfering this IPMK-Sam68 binding interaction with IPMK dominant-negative peptides impaired PLCγ1 phosphorylation.ConclusionsOur results demonstrate that IPMK non-catalytically promotes PLCγ1 phosphorylation by stabilizing the PLCγ1–Sam68 complex. Targeting IPMK in CD4+ T cells may be a promising strategy for managing immune diseases caused by excessive stimulation of TCR.

HECT domain containing-3 (HECTD3) E3 ubiquitin ligase attenuates cardiac ischemia and hypertrophy in mice

Abstract HECT domain containing 3 (HECTD3) is one of the E3 ubiquitin ligases, the cardiac function of which is not completely understood yet. We aimed here to understand the role of HECTD3 in cardiomyocytes using loss- and gain-of-function approaches in in vitro and in vivo models. Interestingly, we found that HECTD3 was significantly downregulated in cardiac tissue obtained from human patients suffering from cardiac hypertrophy and ischemia. We thus employed loss- and gain-of-function approaches in neonatal rat cardiomyocytes (NRVCMs) to get further insights into potential cardiac function of HECTD3. Overexpression of HECTD3 in NRVCMs attenuated cardiomyocyte hypertrophy and lipopolysaccharide (LPS) or interferon-γ (IFN- γ) induced cellular inflammation; its knockdown on the other hand aggravated these effects. Mechanistically, using Yeast two-hybrid screen, mass-spectrometry, and transcriptomics analyses, we identified Small Ubiquitin like Modifier 2 (SUMO2) Signal Transducer and Activator of Transcription-1 (STAT1) as novel substrates for HECTD3, regulation of which resulted into attenuation of hypertrophy and inflammation, respectively. To translate these in vitro results in vivo, we employed Adeno-associated virus-9 (AAV-9) mediated gene therapy approach to overexpress HECTD3 specifically in the heart of mouse models of cardiac ischemia due to permanent ligation of the left anterior descending artery (LAD), and cardiac hypertrophy due to transverse aortic constriction (TAC). Overexpression of HECTD3 in mice, though moderate but significantly improved cardiac function upon ischemic or hypertrophic stress compared to respective control groups. Importantly, in vitro findings were consistent in vivo, where AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, improved cardiac function as assessed by echocardiography, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after TAC or LAD ligation. This data indicates that HECTD3 is a novel modulator of cardiac ischemia, hypertrophy, and inflammation via regulation of SUMO2 and STAT1-signaling. In conclusion, we report here a novel cardioprotective mechanism involving the E3 ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its SUMOylating target STAT1. We believe that this "dual pathway" inhibition exhibits translational importance and could further be exploited for the prevention and/or therapy of heart failure due to cardiac ischemia or hypertrophy.