scholarly journals Transcription Activator Swi6 Interacts with Mbp1 in MluI Cell Cycle Box-Binding Complex and Regulates Hyphal Differentiation and Virulence in Beauveria bassiana

2021 ◽  
Vol 7 (6) ◽  
pp. 411
Author(s):  
Jin-Li Ding ◽  
Jia Hou ◽  
Xiu-Hui Li ◽  
Ming-Guang Feng ◽  
Sheng-Hua Ying
Keyword(s):  
Cell Cycle ◽  
Direct Interaction ◽  
Hyphal Growth ◽  
Transcription Activator ◽  
Liquid Media ◽  
Yeast Two Hybrid ◽  
Genetic Pathways ◽  
Morphological Transitions ◽  
Lethal Time ◽  
Two Hybrid

Mbp1 protein acts as a DNA-binding protein in MluI cell cycle box-binding complex (MBF) and plays an essential role in filamentous myco-pathogen Beauveria bassiana.In the current study, BbSwi6 (a homologue of yeast Swi6) was functionally characterized in B.bassiana. Both BbSwi6 and BbMbp1 localize in the nucleus and display a direct interaction relationship which is indicated by a yeast two-hybrid assay. BbSwi6 significantly contributes to hyphal growth, asexual sporulation and virulence. On the aerial surface, ΔBbSwi6 grew slower on various nutrients and displayed abnormal conidia-producing structures, which hardly produced conidia. In liquid media, BbSwi6 loss led to 90% reduction in blastospore yield. Finally, the virulence of the ΔBbSwi6 mutant was modestly weakened with a reduction of 20% in median lethal time. Comparative transcriptomics revealed that BbSwi6 mediated different transcriptomes during fungal development into conidia and blastospores. Notably, under the indicated condition, the BbSwi6-mediated transcriptome significantly differed to that mediated by BbMbp1. Our results demonstrate that, in addition to their roles as the interactive components in MBF, BbSwi6 and BbMbp1 mediate divergent genetic pathways during morphological transitions in B. bassiana.

scholarly journals The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg–HCPro and VPg–VPg interactions

2003 ◽  
Vol 84 (10) ◽  
pp. 2861-2869 ◽  
Author(s):  
Ma. Leonora M. Yambao ◽  
Chikara Masuta ◽  
Kenji Nakahara ◽  
Ichiro Uyeda
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Yellow Vein ◽  
Transcription Activator ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Clover Yellow Vein Virus ◽  
Two Hybrid ◽  
Far Western Blot

Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV. In addition, a strong HCPro–VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro–VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro–VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg–VPg interaction and the central 19 amino acids were needed for the HCPro–VPg interaction.

scholarly journals The Rotavirus Enterotoxin NSP4 Directly Interacts with the Caveolar Structural Protein Caveolin-1

2006 ◽  
Vol 80 (6) ◽  
pp. 2842-2854 ◽  
Author(s):  
Rebecca D. Parr ◽  
Stephen M. Storey ◽  
DeAnne M. Mitchell ◽  
Avery L. McIntosh ◽  
Minglong Zhou ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Laser Scanning ◽  
Nonstructural Protein ◽  
Direct Interaction ◽  
Cell Periphery ◽  
Structural Protein ◽  
Yeast Two Hybrid ◽  
Ussing Chambers ◽  
Caveolin 1 ◽  
Two Hybrid

ABSTRACT Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4114-135) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast two-hybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.

N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan

2009 ◽  
Vol 420 (3) ◽  
pp. 413-420 ◽  
Author(s):  
Eriko Aoyama ◽  
Takako Hattori ◽  
Mitsuhiro Hoshijima ◽  
Daisuke Araki ◽  
Takashi Nishida ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Connective Tissue ◽  
Connective Tissue Growth Factor ◽  
Binding Protein ◽  
Direct Interaction ◽  
Tissue Growth ◽  
Yeast Two Hybrid ◽  
Ccn Family ◽  
Two Hybrid

CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, as a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type 1 repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan, and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding In vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP–rVWC peptide effectively enhanced the production and release of aggrecan compared with the rTSP–rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC modules, and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.

scholarly journals Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit, Requires Complex Formation of β4 and HD1/Plectin, and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

1998 ◽  
Vol 142 (1) ◽  
pp. 271-284 ◽  
Author(s):  
Roel Q.J. Schaapveld ◽  
Luca Borradori ◽  
Dirk Geerts ◽  
Manuel R. van Leusden ◽  
Ingrid Kuikman ◽  
...  
Keyword(s):  
Bullous Pemphigoid ◽  
Direct Interaction ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Fibronectin Type Iii ◽  
Activation Motif ◽  
Β4 Integrin ◽  
Two Hybrid

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.

scholarly journals Rice Grassy Stunt Tenuivirus Nonstructural Protein p5 Interacts with Itself To Form Oligomeric Complexes In Vitro and In Vivo

2003 ◽  
Vol 77 (1) ◽  
pp. 769-775 ◽  
Author(s):  
Pritsana Chomchan ◽  
Shi-Fang Li ◽  
Yukio Shirako
Keyword(s):  
Hybrid System ◽  
Nonstructural Protein ◽  
Transcription Activator ◽  
Yeast Two Hybrid ◽  
Western Blots ◽  
Yeast Two Hybrid System ◽  
Rice Tissue ◽  
Two Hybrid

ABSTRACT We investigated the interaction of Rice grassy stunt tenuivirus (RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).

Proteasomal interactors control activities as diverse as the cell cycle and glutaminergic neurotransmission

2003 ◽  
Vol 31 (2) ◽  
pp. 470-473 ◽  
Author(s):  
K. Rezvani ◽  
M. Mee ◽  
S. Dawson ◽  
J. McIlhinney ◽  
J. Fujita ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Metabotropic Glutamate Receptors ◽  
Carcinoma Cells ◽  
Genetic Screens ◽  
Yeast Two Hybrid ◽  
Metabotropic Glutamate ◽  
Biological Studies ◽  
E2f Transcription Factor ◽  
Biochemical Analyses ◽  
Two Hybrid

The six regulatory non-redundant ATPases in the base of the 19 S regulator of the 26 S proteasome belong to the AAA superfamily of ATPases. Yeast two-hybrid genetic screens, biochemical analyses and cell biological studies have identified and characterized new interactors of the human S6 (rpt3) and S8 (rpt6) ATPases of the 19 S regulator of the 26 S proteasome. The S6 ATPase interacts with gankyrin. This protein is found in purified human 26 S proteasomes and in a smaller complex(es) containing CDK4 and free S6 ATPase. Gankyrin overexpression causes the phosphorylation of the retinoblastoma protein (pRb) and the release of E2F transcription factor to trigger the expression of DNA synthesis genes. Gankyrin is oncogenic in nude mice and is overexpressed in hepatocellular carcinoma cells (HCCs). The S8 ATPase interacts with members of the large Homer-3 protein family. There are three Homer genes; the Homer 1 and 2 gene products control trafficking and calcium-store-related functions of metabotropic glutamate receptors (e.g. mGluR1α). Homer-3A11 by binding to the S8 ATPase brings mGluR1α to the 26 S proteasome for degradation. The degradation of mGluR1α is blocked by proteasomal inhibitors and by overexpression of the N-terminus of Homer which binds to the receptor. The S8 ATPase and mGluR1α are co-localized in Purkinje dendrites in rat cerebellum. The data are discussed in terms of the regulation of the cell cycle and glutaminergic receptor functions by the 26 S proteasome.

scholarly journals Direct interaction of β-dystroglycan with F-actin

2003 ◽  
Vol 375 (2) ◽  
pp. 329-337 ◽  
Author(s):  
Yun-Ju CHEN ◽  
Heather J. SPENCE ◽  
Jacqueline M. CAMERON ◽  
Thomas JESS ◽  
Jane L. ILSLEY ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Direct Interaction ◽  
Cytoplasmic Tail ◽  
Yeast Two Hybrid ◽  
Glycoprotein Complex ◽  
Biological Studies ◽  
Dystrophin Glycoprotein Complex ◽  
Dystrophin Glycoprotein ◽  
Two Hybrid ◽  
Sarcolemmal Integrity

Dystroglycans are essential transmembrane adhesion receptors for laminin. α-Dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of β-dystroglycan. β-Dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin–glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of β-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells.

scholarly journals Purification of the RelB and RelE Proteins ofEscherichia coli: RelE Binds to RelB and to Ribosomes

2001 ◽  
Vol 183 (8) ◽  
pp. 2700-2703 ◽  
Author(s):  
Cheryl Galvani ◽  
Jefferson Terry ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Direct Interaction ◽  
Binding Activity ◽  
In Vitro Assay ◽  
Ofescherichia Coli ◽  
Yeast Two Hybrid ◽  
Vitro Assay ◽  
Ribosome Binding ◽  
Two Hybrid

ABSTRACT The direct interaction of the Escherichia colicytotoxin RelE with its specific antidote, RelB, was demonstrated in two ways: (i) copurification of the two proteins and (ii) a positive yeast two-hybrid assay involving the relB andrelE genes. In addition, the purified RelE protein exhibited ribosome-binding activity in an in vitro assay, supporting previous observations suggesting that it is an inhibitor of translation.

scholarly journals Rsc3K, a SMV resistance gene localized on chromosome 2 in soybean cv. Kefeng No.1, interacts with virulence determinant P3

2022 ◽  
Author(s):  
Tongtong Jin ◽  
Jinlong Yin ◽  
Song Xue ◽  
Bowen Li ◽  
Tingxuan Zong ◽  
...  
Keyword(s):  
Soybean Mosaic Virus ◽  
Direct Interaction ◽  
Chromosome 2 ◽  
Internal Deletion ◽  
Yeast Two Hybrid ◽  
Viral Pathogens ◽  
Virulence Determinant ◽  
Glycine Max L ◽  
Two Hybrid ◽  
Avirulent Isolate

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens in Glycine max (L.) Merr (soybean). Twenty-two SMV strains (SC1-SC22) isolated in China were identified based on their responses to ten soybean cultivars. By using the F2-derived F3 (F2:3) and recombinant inbred line (RIL) populations of resistant Soybean cultivar (cv.) Kefeng No.1 × susceptible cv. Nannong 1138-2, we localized the gene mediating resistant to SMV-SC3 strain to a 90 kb interval on chromosome 2 in Kefeng No.1. Bean pod mottle vi-rus (BPMV)-induced gene silencing (VIGS) were used to study the gene function of candidate genes in the mapping interval and revealed that an recombinant gene, later named as Rsc3K, caused by internal deletion of a genomic DNA fragement in Kefeng No.1, is the resistant gene to SMV-SC3. By shuffling genes between avirulent isolate SC3 and avirulent SMV isolate 1129, we found that P3 is the virulence determinant causing resistance on Kefeng No.1. We showed the interaction between Rsc3K and P3 by the yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays. In conclusion, this study demonstrated that Rsc3K plays a crucial role in resistance of Kefeng No.1 to SMV-SC3 by direct interaction with viral protein P3.

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