Identification of antituberculosis agents that target ribosomal protein interactions using a yeast two-hybrid system

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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A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to In cellula Affinities

Molecular & Cellular Proteomics ◽

10.1074/mcp.tir119.001692 ◽

2020 ◽

Vol 19 (4) ◽

pp. 701-715 ◽

Cited By ~ 1

Author(s):

David Cluet ◽

Ikram Amri ◽

Blandine Vergier ◽

Jérémie Léault ◽

Astrid Audibert ◽

...

Keyword(s):

Flow Cytometry ◽

Hybrid System ◽

Protein Interactions ◽

Dissociation Constants ◽

Automated Analysis ◽

Technological Advancement ◽

Yeast Two Hybrid ◽

Expression Levels ◽

Yeast Two Hybrid System ◽

Two Hybrid

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm. After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

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The yeast two-hybrid system for studying protein—protein interactions

Current Opinion in Biotechnology ◽

10.1016/0958-1669(95)80010-7 ◽

1995 ◽

Vol 6 (1) ◽

pp. 59-64 ◽

Cited By ~ 52

Author(s):

Jeremy Luban ◽

Stephen P Goff

Keyword(s):

Hybrid System ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

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Neurofilament triplet protein interactions: evidence for the preferred formation of NF-L-containing dimers and a putative function for the end domains

Journal of Cell Science ◽

10.1242/jcs.109.10.2493 ◽

1996 ◽

Vol 109 (10) ◽

pp. 2493-2498 ◽

Cited By ~ 3

Author(s):

D.A. Carpenter ◽

W. Ip

Keyword(s):

Intermediate Filaments ◽

Hybrid System ◽

Weak Interaction ◽

Molecular Interactions ◽

Protein Interactions ◽

Building Blocks ◽

Full Length ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

In this report we examine the molecular interactions that lead to formation of neurofilaments, the intermediate filaments in neurons. Using the yeast two-hybrid system, we found that the rod domains of all three NF triplet proteins interacted strongly with one another and with rod domains of the Type III IF proteins, vimentin and desmin. A slight preference toward NF-L-containing dimers was observed over ones not containing NF-L. Interactions among the full length NF triplet proteins exhibited more specificity. Full length NF-L had only a relatively weak interaction with another full length NF-L molecule, but reacted more robustly with full length NF-M or NF-H lacking only part of the head domain. No homologous or heterologous dimerization of NF-M and NF-H was detectable. These results support the hypothesis that neurofilaments are obligate heteropolymers and that heterodimeric subunits are the preferred building blocks. They further suggest that the mechanism that specifies heterodimeric interaction among the NF triplet proteins resides in the end domains.

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