Yeast two-hybrid screens imply involvement of fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport

Here, the constituents of coffee with estrogenic activity are summarized by a comprehensive literature search, and their mechanisms of action for their physiological effects are discussed at the molecular and cellular levels. The estrogenic activity of coffee constituents, such as acids, caramelized products, carbohydrates, lignin, minerals, nitrogenous compounds, oil (lipids), and others, such as volatile compounds, was first evaluated by activity assays, such as animal tests, cell assay, ligand-binding assay, protein assay, reporter-gene assay, transcription assay, and yeast two-hybrid assay. Second, the health benefits associated with the estrogenic coffee constituents, such as bone protection, cancer treatment/prevention, cardioprotection, neuroprotection, and the improvement of menopausal syndromes, were summarized, including their potential therapeutic/clinical applications. Inconsistent results regarding mixed estrogenic/anti-estrogenic/non-estrogenic or biphasic activity, and unbeneficial effects associated with the constituents, such as endocrine disruption, increase the complexity of the effects of estrogenic coffee constituents. However, as the increase of the knowledge about estrogenic cell signaling, such as the types of specific signaling pathways, selective modulations of cell signaling, signal crosstalk, and intercellular/intracellular networks, pathway-based assessment will become a more realistic means in the future to more reliably evaluate the beneficial applications of estrogenic coffee constituents.

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Investigation of Fanconi Anemia Protein Interactions by Yeast Two-Hybrid Analysis

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1999.2055 ◽

2000 ◽

Vol 268 (1) ◽

pp. 73-77 ◽

Cited By ~ 13

Author(s):

Pia A.J. Huber ◽

Annette L. Medhurst ◽

Hagop Youssoufian ◽

Christopher G. Mathew

Keyword(s):

Fanconi Anemia ◽

Protein Interactions ◽

Yeast Two Hybrid ◽

Hybrid Analysis ◽

Two Hybrid

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FANCC, FANCE, and FANCD2 Form a Ternary Complex Essential to the Integrity of the Fanconi Anemia DNA Damage Response Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m507758200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36118-36125 ◽

Cited By ~ 30

Author(s):

Susan M. Gordon ◽

Noa Alon ◽

Manuel Buchwald

Keyword(s):

Hybrid System ◽

Fanconi Anemia ◽

Protein Complex ◽

Bone Marrow Failure ◽

Critical Role ◽

Yeast Two Hybrid ◽

Complex Activity ◽

Yeast Two Hybrid System ◽

Two Hybrid ◽

Complex Components

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, cancer predisposition, and increased cellular sensitivity to DNA-cross-linking agents. The products of seven of the nine identified FA genes participate in a protein complex required for monoubiquitination of the FANCD2 protein. Direct interaction of the FANCE protein with both fellow FA complex component FANCC and the downstream FANCD2 protein has been observed in the yeast two-hybrid system. Here, we demonstrate the ability of FANCE to mediate the interaction between FANCC and FANCD2 in the yeast three-hybrid system and confirm the FANCE-mediated association of FANCC with FANCD2 in human cells. A yeast two-hybrid system-based screen was devised to identify randomly mutagenized FANCE proteins capable of interaction with FANCC but not with FANCD2. Exogenous expression of these mutants in an FA-E cell line and subsequent evaluation of FANCD2 monoubiquitination and DNA cross-linker sensitivity indicated a critical role for the FANCE/FANCD2 interaction in maintaining FA pathway integrity. Three-hybrid experiments also demonstrated the ability of FANCE to mediate the interaction between FA core complex components FANCC and FANCF, indicating an additional role for FANCE in complex assembly. Thus, FANCE is shown to be a key mediator of protein interactions both in the architecture of the FA protein complex and in the connection of complex components to the putative downstream targets of complex activity.

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Faculty Opinions recommendation of A New Method, "Reverse Yeast Two-Hybrid Array" (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727593819.793532978 ◽

2017 ◽

Author(s):

Anuj Kumar ◽

Amberlene De La Rocha

Keyword(s):

New Method ◽

Physical Interaction ◽

Yeast Two Hybrid ◽

Two Hybrid

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Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

ACTA AGRONOMICA SINICA ◽

10.3724/sp.j.1006.2012.01583 ◽

2013 ◽

Vol 38 (9) ◽

pp. 1583-1591

Author(s):

Li-Yan XUE ◽

Bing LUO ◽

Li-Quan ZHU ◽

Yong-Jun YANG ◽

He-Cui ZHANG ◽

...

Keyword(s):

Hybrid System ◽

Dna Sequences ◽

Extracellular Domain ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

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DETECTION OF MICROCYSTINS BASED ON YEAST TWO-HYBRID SYSTEM

Acta Hydrobiologica Sinica ◽

10.3724/sp.j.1035.2008.00201 ◽

2008 ◽

Vol 32 (2) ◽

pp. 201-206

Author(s):

Xiao PAN

Keyword(s):

Hybrid System ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

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Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

Journal of Virology ◽

10.1128/jvi.01642-19 ◽

2019 ◽

Vol 94 (1) ◽

Cited By ~ 3

Author(s):

M. V. Borca ◽

E. A. Vuono ◽

E. Ramirez-Medina ◽

P. Azzinaro ◽

K. A. Berggren ◽

...

Keyword(s):

Amino Acid ◽

Protein Interaction ◽

Hybrid Approach ◽

Classical Swine Fever ◽

Host Protein ◽

Yeast Two Hybrid ◽

Protein Protein Interaction ◽

Viral Virulence ◽

Glycoprotein E2 ◽

Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

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