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1.
Gen Physiol Biophys ; 43(4): 367-370, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38953578

ABSTRACT

Alzheimer's disease is currently not curable. Almost all attempts to identify disease-modifying drugs failed and the causes of disease etiology are not well understood. Neurofibrillary tangles composed of pathological tau protein belong to the main hallmarks of this disease. Identification of novel physiological and pathological tau interacting proteins may lead to a better understanding of Alzheimer's disease pathology and tau physiology and therefore we performed a screening of the brain library by a yeast two-hybrid system intending to identify new tau interaction partners. We identified CHORDC1 (cysteine and histidine-rich domain-containing protein 1) as a novel tau interaction partner by this approach. The CHORDC1-tau interaction was validated by co-immunoprecipitation from rat brain tissues and by in vitro co-localization in the cellular model expressing full-length human tau protein. We believe that our results can be useful for researchers studying tau protein in health and disease.


Subject(s)
tau Proteins , tau Proteins/metabolism , Rats , Animals , Humans , Protein Binding , Brain/metabolism , Protein Interaction Mapping , Two-Hybrid System Techniques
2.
PLoS Pathog ; 20(7): e1012411, 2024 Jul.
Article in English | MEDLINE | ID: mdl-39024411

ABSTRACT

Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T. gondii. In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0's localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701-877 of BCC0 are essential for both its localization and function and that residues 1-899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0's essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.


Subject(s)
Protozoan Proteins , Toxoplasma , Toxoplasma/metabolism , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Cell Division , Two-Hybrid System Techniques , Membrane Proteins/metabolism , Membrane Proteins/genetics
3.
Physiol Plant ; 176(4): e14451, 2024.
Article in English | MEDLINE | ID: mdl-39075941

ABSTRACT

The regulation of fruit development is a complex process and a core issue in the fruit tree industry. To investigate the role of PbGIF1 in pear fruit development, we identified a transcription factor PbbHLH137 that regulates pear (Pyrus bretschneideri) fruit development by screening a yeast library constructed from fruit cDNA. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and split luciferase complementation (split-LUC) assays were performed to confirm the PbbHLH137-PbGIF1 interaction. By tracing the complete fruit development process, we found that PbbHLH137 expression was closely related to fruit size and highly involved at the late pear fruit development stage. Transgenic experiments showed that heterologous expression of PbbHLH137 or PbGIF1 promoted fruit enlargement. PbbHLH137 promoted mainly the expansion of fruit cell volume, whereas PbGIF1 mainly increased the number of cells. Further LUC experiments demonstrated that PbGIF1 promoted the transcriptional activation ability of PbbHLH137. Our work identified PbbHLH137 as a transcription factor that regulates fruit development, and showed that PbGIF1 played an ongoing role during fruit development, making it a candidate gene for genetic improvement of pear fruit development.


Subject(s)
Fruit , Gene Expression Regulation, Plant , Plant Proteins , Pyrus , Transcription Factors , Pyrus/genetics , Pyrus/growth & development , Pyrus/metabolism , Fruit/growth & development , Fruit/genetics , Fruit/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Plants, Genetically Modified , Two-Hybrid System Techniques
4.
Peptides ; 179: 171269, 2024 Sep.
Article in English | MEDLINE | ID: mdl-38960286

ABSTRACT

bZIP transcription factors can function as homodimers or heterodimers through interactions with their disordered coiled-coil domain. Such dimer assemblies are known to influence DNA-binding specificity and/or the recruitment of binding partners, which can cause a functional switch of a transcription factor from being an activator to a repressor. We recently identified the genomic targets of a bZIP transcription factor called CREB3L1 in rat hypothalamic supraoptic nucleus by ChIP-seq. The objective of this study was to investigate the CREB3L1 protein-to-protein interactome of which little is known. For this approach, we created and screened a rat supraoptic nucleus yeast two-hybrid prey library with the bZIP region of rat CREB3L1 as the bait. Our yeast two-hybrid approach captured five putative CREB3L1 interacting prey proteins in the supraoptic nucleus. One interactor was selected by bioinformatic analyses for more detailed investigation by co-immunoprecipitation, immunofluorescent cellular localisation, and reporter assays in vitro. Here we identify dimerisation hub protein Dynein Light Chain LC8-Type 1 as a CREB3L1 interacting protein that in vitro enhances CREB3L1 activation of target genes.


Subject(s)
Cyclic AMP Response Element-Binding Protein , Cytoplasmic Dyneins , Nerve Tissue Proteins , Transcriptional Activation , Animals , Humans , Rats , Arginine Vasopressin/metabolism , Arginine Vasopressin/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Cytoplasmic Dyneins/metabolism , Cytoplasmic Dyneins/genetics , Protein Multimerization , Supraoptic Nucleus/metabolism , Transcriptional Activation/genetics , Two-Hybrid System Techniques
5.
PeerJ ; 12: e17540, 2024.
Article in English | MEDLINE | ID: mdl-38887620

ABSTRACT

Despite extensive research highlighting the pivotal role of MYB transcription factors in regulating anthocyanin biosynthesis, the interactive regulatory network involving these MYB factors in pear fruits remains inadequately characterized. In this study, the anthocyanin-regulatory gene PbrMYB114 was successfully cloned from 'Yuluxiang' pear (Pyrus bretschneideri) fruits, and its influence on anthocyanin accumulation was confirmed through transient expression assays. Specifically, the co-transformation of PbrMYB114 with its partner PbrbHLH3 in pears served to validate the functional role of PbrMYB114. Subsequently, PbrMYB114 was employed as bait in a yeast two-hybrid screening assay, using a 'Yuluxiang' pear protein library, which led to the identification of 25 interacting proteins. Further validation of the interactions between PbrMYB114 and PbrMT2/PbrMT3 was conducted. Investigations into the role of PbrMT2 and PbrMT3 in 'Duli' seedlings (Pyrus betulaefolia) revealed their potential to enhance anthocyanin accumulation. The outcomes of these studies provide novel insights into the protein network that regulates pear anthocyanin biosynthesis, particularly the functional interactions among PbrMYB114 and associated proteins.


Subject(s)
Anthocyanins , Gene Expression Regulation, Plant , Plant Proteins , Pyrus , Transcription Factors , Pyrus/metabolism , Pyrus/genetics , Anthocyanins/metabolism , Anthocyanins/genetics , Anthocyanins/biosynthesis , Transcription Factors/genetics , Transcription Factors/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Two-Hybrid System Techniques , Fruit/metabolism , Fruit/genetics
6.
Mol Syst Biol ; 20(7): 825-844, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38849565

ABSTRACT

Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.


Subject(s)
DNA , PAX6 Transcription Factor , PAX6 Transcription Factor/genetics , PAX6 Transcription Factor/metabolism , Humans , DNA/genetics , DNA/metabolism , Binding Sites , Protein Binding , Mutation , Two-Hybrid System Techniques , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Mutation, Missense , Transcription Factors/genetics , Transcription Factors/metabolism , DNA Mutational Analysis
7.
J Plant Physiol ; 300: 154299, 2024 Sep.
Article in English | MEDLINE | ID: mdl-38936241

ABSTRACT

The F-box protein (FBP) family plays diverse functions in the plant kingdom, with the function of many members still unrevealed. In this study, a specific FBP called PmFBK2, containing Kelch repeats from Persicaria minor, was functionally investigated. Employing the yeast two-hybrid (Y2H) assay, PmFBK2 was found to interact with Skp1-like proteins from P. minor, suggesting its potential to form an E3 ubiquitin ligase, known as the SCF complex. Y2H and co-immunoprecipitation tests revealed that PmFBK2 interacts with full-length PmGID1b. The interaction marks the first documented binding between these two protein types, which have never been reported in other plants before, and they exhibited a negative effect on gibberellin (GA) signal transduction. The overexpression of PmFBK2 in the kmd3 mutant, a homolog from Arabidopsis, demonstrated the ability of PmFBK2 to restore the function of the mutated KMD3 gene. The function restoration was supported by morphophysiological and gene expression analyses, which exhibited patterns similar to the wild type (WT) compared to the kmd3 mutant. Interestingly, the overexpression of PmFBK2 or PmGID1b in Arabidopsis had opposite effects on rosette diameter, seed weight, and plant height. This study provides new insights into the complex GA signalling. It highlights the crucial roles of the interaction between FBP and the GA receptor (GID1b) in regulating GA responses. These findings have implications for developing strategies to enhance plant growth and yield by modulating GA signalling in crops.


Subject(s)
F-Box Proteins , Gibberellins , Plant Proteins , Signal Transduction , Gibberellins/metabolism , F-Box Proteins/metabolism , F-Box Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/genetics , Gene Expression Regulation, Plant , Two-Hybrid System Techniques , Plant Growth Regulators/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism
8.
Plant Mol Biol ; 114(3): 56, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38743198

ABSTRACT

Most eukaryotic organisms employ a telomerase complex for the maintenance of chromosome ends. The core of this complex is composed of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) subunits. The TERT reverse transcriptase (RT) domain synthesises telomeric DNA using the TR template sequence. The other TERT domains contribute to this process in different ways. In particular, the TERT RNA-binding domain (TRBD) interacts with specific TR motif(s). Using a yeast 3-hybrid system, we show the critical role of Arabidopsis thaliana (At) TRBD and embryophyta-conserved KRxR motif in the unstructured linker preceding the TRBD domain for binding to the recently identified AtTR subunit. We also show the essential role of the predicted P4 stem and pseudoknot AtTR structures and provide evidence for the binding of AtTRBD to pseudoknot and KRxR motif stabilising interaction with the P4 stem structure. Our results thus provide the first insight into the core part of the plant telomerase complex.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Telomerase , Telomerase/genetics , Telomerase/metabolism , Telomerase/chemistry , Arabidopsis/genetics , Arabidopsis/enzymology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/chemistry , RNA/metabolism , RNA/genetics , Two-Hybrid System Techniques , RNA, Plant/genetics , RNA, Plant/metabolism , Nucleic Acid Conformation , Protein Binding
9.
Int J Mol Sci ; 25(10)2024 May 16.
Article in English | MEDLINE | ID: mdl-38791467

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Two-Hybrid System Techniques , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , PII Nitrogen Regulatory Proteins/metabolism , PII Nitrogen Regulatory Proteins/genetics , Cyanobacteria/metabolism , Cyanobacteria/genetics , Escherichia coli/metabolism , Escherichia coli/genetics , Protein Binding
10.
Methods Mol Biol ; 2808: 89-103, 2024.
Article in English | MEDLINE | ID: mdl-38743364

ABSTRACT

The study of virus-host interactions is essential to achieve a comprehensive understanding of the viral replication process. The commonly used methods are yeast two-hybrid approach and transient expression of a single tagged viral protein in host cells followed by affinity purification of interacting cellular proteins and mass spectrometry analysis (AP-MS). However, by these approaches, virus-host protein-protein interactions are detected in the absence of a real infection, not always correctly compartmentalized, and for the yeast two-hybrid approach performed in a heterologous system. Thus, some of the detected protein-protein interactions may be artificial. Here we describe a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect protein partners during the infection (AP-MS in viral context). This way, virus-host protein-protein interacting co-complexes can be purified directly from infected cells for further characterization.


Subject(s)
Host-Pathogen Interactions , Measles virus , Reverse Genetics , Viral Proteins , Measles virus/genetics , Humans , Host-Pathogen Interactions/genetics , Reverse Genetics/methods , Viral Proteins/metabolism , Viral Proteins/genetics , Two-Hybrid System Techniques , Virus Replication , Mass Spectrometry , Protein Interaction Mapping/methods , Measles/virology , Measles/metabolism , Animals , Protein Binding
11.
Plant Signal Behav ; 19(1): 2353536, 2024 Dec 31.
Article in English | MEDLINE | ID: mdl-38771929

ABSTRACT

Cellular behavior, cell differentiation and ontogenetic development in eukaryotes result from complex interactions between epigenetic and classic molecular genetic mechanisms, with many of these interactions still to be elucidated. Histone deacetylase enzymes (HDACs) promote the interaction of histones with DNA by compacting the nucleosome, thus causing transcriptional repression. MADS-domain transcription factors are highly conserved in eukaryotes and participate in controlling diverse developmental processes in animals and plants, as well as regulating stress responses in plants. In this work, we focused on finding out putative interactions of Arabidopsis thaliana HDACs and MADS-domain proteins using an evolutionary perspective combined with bioinformatics analyses and testing the more promising predicted interactions through classic molecular biology tools. Through bioinformatic analyses, we found similarities between HDACs proteins from different organisms, which allowed us to predict a putative protein-protein interaction between the Arabidopsis thaliana deacetylase HDA15 and the MADS-domain protein XAANTAL1 (XAL1). The results of two-hybrid and Bimolecular Fluorescence Complementation analysis demonstrated in vitro and in vivo HDA15-XAL1 interaction in the nucleus. Likely, this interaction might regulate developmental processes in plants as is the case for this type of interaction in animals.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Histone Deacetylases , MADS Domain Proteins , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Histone Deacetylases/metabolism , Histone Deacetylases/genetics , MADS Domain Proteins/metabolism , MADS Domain Proteins/genetics , Protein Binding , Two-Hybrid System Techniques
12.
Int J Biol Macromol ; 268(Pt 1): 131704, 2024 May.
Article in English | MEDLINE | ID: mdl-38670198

ABSTRACT

Mosquitoes form a vital group of vector insects, which can transmit various diseases and filarial worms. The cuticle is a critical structure that protects mosquitoes from adverse environmental conditions and penetration resistance. Thus, cuticle proteins can be used as potential targets for controlling the mosquito population. In the present study, we found that AaCPR100A is a structural protein in the soft cuticle, which has flexibility and elasticity allowing insects to move or fly freely, of Aedes aegypti. RNA interference (RNAi) of AaCPR100A caused high mortality in Aedes aegypti larvae and adults and significantly decreased the egg hatching rate. Transmission electron microscopy (TEM) analysis revealed that the larval microstructure had no recognizable endocuticle in AaCPR100A-deficient mosquitoes. A yeast two-hybrid assay was performed to screen proteins interacting with AaCPR100A. We verified that the G12-like protein had the strongest interaction with AaCPR100A using yeast two-hybrid and GST pull-down assays. Knockdown of G12-like transcription resulted in high mortality in Ae. aegypti larvae, but not in adults. Interestingly, RNAi of G12-like rescued the high mortality of adults caused by decreased AaCPR100A expression. Additionally, adults treated with G12-like dsRNA were found to be sensitive to low temperature, and their eggshell formation and hatching were decreased. Overall, our results demonstrated that G12-like may interacts with AaCPR100A, and both G12-like and AaCPR100A are involved in Ae. aegypti cuticle development and eggshell formation. AaCPR100A and G12-like can thus be considered newly potential targets for controlling the Ae. aegypti mosquito.


Subject(s)
Aedes , Insect Proteins , Animals , Aedes/genetics , Aedes/metabolism , Insect Proteins/genetics , Insect Proteins/metabolism , Larva/metabolism , Larva/growth & development , RNA Interference , Protein Binding , Two-Hybrid System Techniques
13.
Planta ; 259(5): 122, 2024 Apr 15.
Article in English | MEDLINE | ID: mdl-38619628

ABSTRACT

MAIN CONCLUSION: Overexpression of BnaC02.TPS8 increased low N and high sucrose-induced anthocyanin accumulation. Anthocyanin plays a crucial role in safeguarding photosynthetic tissues against high light, UV radiation, and oxidative stress. Their accumulation is triggered by low nitrogen (N) stress and elevated sucrose levels in Arabidopsis. Trehalose-6-phosphate (T6P) serves as a pivotal signaling molecule, sensing sucrose availability, and carbon (C) metabolism. However, the mechanisms governing the regulation of T6P synthase (TPS) genes responsible for anthocyanin accumulation under conditions of low N and high sucrose remain elusive. In a previous study, we demonstrated the positive impact of a cytoplasm-localized class II TPS protein 'BnaC02.TPS8' on photosynthesis and seed yield improvement in Brassica napus. The present research delves into the biological role of BnaC02.TPS8 in response to low N and high sucrose. Ectopic overexpression of BnaC02.TPS8 in Arabidopsis seedlings resulted in elevated shoot T6P levels under N-sufficient conditions, as well as an increased carbon-to-nitrogen (C/N) ratio, sucrose accumulation, and starch storage under low N conditions. Overexpression of BnaC02.TPS8 in Arabidopsis heightened sensitivity to low N stress and high sucrose levels, accompanied by increased anthocyanin accumulation and upregulation of genes involved in flavonoid biosynthesis and regulation. Metabolic profiling revealed increased levels of intermediate products of carbon metabolism, as well as anthocyanin and flavonoid derivatives in BnaC02.TPS8-overexpressing Arabidopsis plants under low N conditions. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses demonstrated that BnaC02.TPS8 interacts with both BnaC08.TPS9 and BnaA01.TPS10. These findings contribute to our understanding of how TPS8-mediated anthocyanin accumulation is modulated under low N and high sucrose conditions.


Subject(s)
Arabidopsis , Brassica napus , Sugar Phosphates , Trehalose , Anthocyanins , Arabidopsis/genetics , Brassica napus/genetics , Carbon , Flavonoids , Nitrogen , Trehalose/analogs & derivatives , Two-Hybrid System Techniques
14.
Plant Physiol Biochem ; 210: 108600, 2024 May.
Article in English | MEDLINE | ID: mdl-38593488

ABSTRACT

Populus euphratica phospholipase Dδ (PePLDδ) is transcriptionally regulated and mediates reactive oxygen species (ROS) and ion homeostasis under saline conditions. The purpose of this study is to explore the post-transcriptional regulation of PePLDδ in response to salt environment. P. euphratica PePLDδ was shown to interact with the NADP-dependent malic enzyme (NADP-ME) by screening the yeast two-hybrid libraries. The transcription level of PeNADP-ME increased upon salt exposure to NaCl (200 mM) in leaves and roots of P. euphratica. PeNADP-ME had a similar subcellular location with PePLDδ in the cytoplasm, and the interaction between PeNADP-ME and PePLDδ was further verified by GST pull-down and yeast two-hybrid. To clarify whether PeNADP-ME interacts with PePLDδ to enhance salt tolerance, PePLDδ and PeNADP-ME were overexpressed singly or doubly in Arabidopsis thaliana. Dual overexpression of PeNADP-ME and PePLDδ resulted in an even more pronounced improvement in salt tolerance compared with single transformants overexpressing PeNADP-ME or PePLDδ alone. Greater Na+ limitation and Na+ efflux in roots were observed in doubly overexpressed plants compared with singly overexpressed plants with PeNADP-ME or PePLDδ. Furthermore, NaCl stimulation of SOD, APX, and POD activity and transcription were more remarkable in the doubly overexpressed plants. It is noteworthy that the enzymic activity of NADP-ME and PLD, and total phosphatidic acid (PA) concentrations were significantly higher in the double-overexpressed plants than in the single transformants. We conclude that PeNADP-ME interacts with PePLDδ in Arabidopsis to promote PLD-derived PA signaling, conferring Na+ extrusion and ROS scavenging under salt stress.


Subject(s)
Homeostasis , Phospholipase D , Plant Proteins , Populus , Salt Stress , Arabidopsis/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant/drug effects , Phospholipase D/metabolism , Phospholipase D/genetics , Plant Proteins/metabolism , Plant Proteins/genetics , Plant Roots/metabolism , Plant Roots/genetics , Plant Roots/drug effects , Plants, Genetically Modified , Populus/metabolism , Populus/genetics , Populus/drug effects , Reactive Oxygen Species/metabolism , Salt Stress/genetics , Salt Tolerance/genetics , Sodium Chloride/pharmacology , Two-Hybrid System Techniques
15.
Biomolecules ; 14(4)2024 Mar 24.
Article in English | MEDLINE | ID: mdl-38672406

ABSTRACT

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.


Subject(s)
Nitric Oxide Synthase Type II , Testis , Two-Hybrid System Techniques , Humans , Male , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type II/genetics , Testis/metabolism , HEK293 Cells , Protein Binding
16.
Cells ; 13(8)2024 Apr 17.
Article in English | MEDLINE | ID: mdl-38667311

ABSTRACT

Actin is a protein of central importance to many cellular functions. Its localization and activity are regulated by interactions with a high number of actin-binding proteins. In a yeast two-hybrid (Y2H) screening system, snail family transcriptional repressor 2 (SNAI2 or slug) was identified as a yet unknown potential actin-binding protein. We validated this interaction using immunoprecipitation and analyzed the functional relation between slug and actin. Since both proteins have been reported to be involved in DNA double-strand break (DSB) repair, we focused on their interaction during this process after treatment with doxorubicin or UV irradiation. Confocal microscopy elicits that the overexpression of actin fused to an NLS stabilizes complexes of slug and γH2AX, an early marker of DNA damage repair.


Subject(s)
Actins , Protein Binding , Snail Family Transcription Factors , Snail Family Transcription Factors/metabolism , Snail Family Transcription Factors/genetics , Actins/metabolism , Humans , Cell Nucleus/metabolism , Histones/metabolism , Two-Hybrid System Techniques , DNA Repair , Doxorubicin/pharmacology , DNA Breaks, Double-Stranded , Ultraviolet Rays , Animals
17.
J Biol Chem ; 300(3): 105741, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38340793

ABSTRACT

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.


Subject(s)
Bacterial Proteins , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa , Transcription, Genetic , Type VI Secretion Systems , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Electrophoretic Mobility Shift Assay , Immunoprecipitation , Mutation , Promoter Regions, Genetic , Protein Binding , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Pyocyanine/metabolism , Quorum Sensing , Second Messenger Systems , Two-Hybrid System Techniques , Type VI Secretion Systems/genetics , Type VI Secretion Systems/metabolism
18.
Acta Parasitol ; 69(1): 505-513, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38206477

ABSTRACT

BACKGROUND: Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a primitive opportunistic protozoon, and one of the earliest differentiated eukaryotes. Despite its primitive nature, G. lamblia has a sophisticated cytoskeleton system, which is closely related to its proliferation and pathogenicity. Meanwhile, α giardin is a G. lamblia-specific cytoskeleton protein, which belongs to the annexin superfamily. Interestingly, G. lamblia has 21 annexin-like α giardins, i.e., more than higher eukaryotes. The functional differences among α giardin members are not fully understood. METHODS: We took α-4 giardin, a member of α giardin family, as a research object. A morpholino-mediated knockdown experiment was performed to identify the effect of α-4 giardin on G. lamblia trophozoites biological traits. A yeast two-hybrid cDNA library of G. lamblia strain C2 trophozoites was screened for interaction partners of α-4 giardin. Co-immunoprecipitation and fluorescent colocalization confirmed the relationship between G. lamblia EB1 (gEB1) and α-4 giardin. RESULTS: α-4 Giardin could inhibit the proliferation and adhesion of G. lamblia trophozoites. In addition, it interacted with G. lamblia EB1 (gEB1). CONCLUSIONS: α-4 Giardin was involved in proliferation and adhesion in G. lamblia trophozoites, and EB1, a crucial roles in mitosis, was an interacting partner of α-4 giardin.


Subject(s)
Cytoskeletal Proteins , Giardia lamblia , Protozoan Proteins , Trophozoites , Giardia lamblia/metabolism , Giardia lamblia/genetics , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Trophozoites/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/genetics , Protein Binding , Two-Hybrid System Techniques
19.
STAR Protoc ; 5(1): 102856, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38285736

ABSTRACT

Analyses of long non-coding RNA (lncRNA)-protein interactions provide key clues for understanding the molecular basis of lncRNA-modulated biological processes. Here, we detail a yeast three-hybrid assay to identify the lncRNA-interacting protein. We describe steps for lncRNA bait preparation, screening an RNA-binding proteins (RBPs) cDNA library, and validation of the lncRNA-RBP interaction. The assay can also be further applied to delineate the region of RBP that mediates the RNA-protein interaction. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.


Subject(s)
RNA, Long Noncoding , Two-Hybrid System Techniques , RNA, Long Noncoding/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Gene Library
20.
J Biol Chem ; 300(1): 105582, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38141762

ABSTRACT

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T. gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an in vivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a Kd of 334 nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T. gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T. gondii fitness.


Subject(s)
GTPase-Activating Proteins , Mitochondria , Protein Interaction Maps , Protozoan Proteins , Toxoplasma , Humans , Binding Sites , Calorimetry , Chromatography, Gel , Fibroblasts/metabolism , Fibroblasts/parasitology , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Hydrogen Deuterium Exchange-Mass Spectrometry , Mitochondria/metabolism , Mitochondria/parasitology , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Toxoplasma/chemistry , Toxoplasma/genetics , Toxoplasma/metabolism , Two-Hybrid System Techniques
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