scholarly journals A yeast two-hybrid smart-pool-array system for protein-interaction mapping

2007 ◽  
Vol 4 (5) ◽  
pp. 405-407 ◽  
Author(s):  
Fulai Jin ◽  
Larisa Avramova ◽  
Jing Huang ◽  
Tony Hazbun
Keyword(s):  
Protein Interaction ◽  
Yeast Two Hybrid ◽  
Two Hybrid

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

2019 ◽  
Vol 94 (1) ◽  
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.

scholarly journals Construction of gateway-compatible yeast two-hybrid vectors for high throughput analysis of protein interaction

2010 ◽  
Vol 9 (9) ◽  
pp. 1392-1396
Author(s):  
Zhu Tingheng ◽  
Wang Weixia ◽  
Wong Hann lin ◽  
Yang Xiao ◽  
Wang Kun ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interaction ◽  
Yeast Two Hybrid ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
Hybrid Vectors ◽  
Two Hybrid

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

scholarly journals Quantifying noise in mass spectrometry and yeast two-hybrid protein interaction detection experiments

2015 ◽  
Vol 12 (110) ◽  
pp. 20150573 ◽  
Author(s):  
A. Annibale ◽  
A. C. C. Coolen ◽  
N. Planell-Morell
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Interaction Networks ◽  
Interaction Networks ◽  
Yeast Two Hybrid ◽  
Binary Matrix ◽  
Interaction Detection ◽  
Experimental Protocols ◽  
True Protein ◽  
Two Hybrid

Protein interaction networks (PINs) are popular means to visualize the proteome. However, PIN datasets are known to be noisy, incomplete and biased by the experimental protocols used to detect protein interactions. This paper aims at understanding the connection between true protein interactions and the protein interaction datasets that have been obtained using the most popular experimental techniques, i.e. mass spectronomy and yeast two-hybrid. We start from the observation that the adjacency matrix of a PIN, i.e. the binary matrix which defines, for every pair of proteins in the network, whether or not there is a link, has a special form, that we call separable. This induces precise relationships between the moments of the degree distribution (i.e. the average number of links that a protein in the network has, its variance, etc.) and the number of short loops (i.e. triangles, squares, etc.) along the links of the network. These relationships provide powerful tools to test the reliability of datasets and hint at the underlying biological mechanism with which proteins and complexes recruit each other.

scholarly journals Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system

2001 ◽  
Vol 82 (4) ◽  
pp. 935-939 ◽  
Author(s):  
Deyin Guo ◽  
Minna-Liisa Rajamäki ◽  
Mart Saarma ◽  
Jari P. T. Valkonen
Keyword(s):  
Hybrid System ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Yeast Two Hybrid ◽  
Potato Virus A ◽  
Yeast Two Hybrid System ◽  
Multiple Protein ◽  
Interaction Map ◽  
Two Hybrid ◽  
Shed Light

A map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus Potyvirus, family Potyviridae) was generated using the yeast two-hybrid system (YTHS). Interactions were readily detected with five PVA protein combinations (HC–HC, HC–CI, VPg–VPg, NIa–NIb and CP–CP) and weak but reproducible interactions were detected for seven additional combinations (P1–CI, P3–NIb, NIaPro–NIb, VPg–NIa, VPg–NIaPro, NIaPro–NIa and NIa–NIa). In PSbMV, readily detectable interactions were found in five protein combinations (HC–HC, VPg–VPg, VPg–NIa, NIa–NIa and NIa–NIb) and weaker but reproducible interactions were detected for three additional combinations (P3–NIa, NIa–NIaPro and CP–CP). The self-interactions of HC, VPg, NIa and CP and the interactions of VPg–NIa, NIa–NIaPro and NIa–NIb were, therefore, common for the two potyviruses. The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.

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