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Target identification strategies in plant chemical biology.

Dejonghe W, Russinova E - Front Plant Sci (2014)

Bottom Line: The current needs to understand gene function in plant biology increasingly require more dynamic and conditional approaches opposed to classic genetic strategies.Gene redundancy and lethality can substantially complicate research, which might be solved by applying a chemical genetics approach.Now understood as the study of small molecules and their effect on biological systems with subsequent target identification, chemical genetics is a fast developing field with a strong history in pharmaceutical research and drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Systems Biology, VIB Ghent, Belgium ; Department of Plant Biotechnology and Bioinformatics, Ghent University Ghent, Belgium.

ABSTRACT
The current needs to understand gene function in plant biology increasingly require more dynamic and conditional approaches opposed to classic genetic strategies. Gene redundancy and lethality can substantially complicate research, which might be solved by applying a chemical genetics approach. Now understood as the study of small molecules and their effect on biological systems with subsequent target identification, chemical genetics is a fast developing field with a strong history in pharmaceutical research and drug discovery. In plant biology however, chemical genetics is still largely in the starting blocks, with most studies relying on forward genetics and phenotypic analysis for target identification, whereas studies including direct target identification are limited. Here, we provide an overview of recent advances in chemical genetics in plant biology with a focus on target identification. Furthermore, we discuss different strategies for direct target identification and the possibilities and challenges for plant biology.

No MeSH data available.


General principle of emerging and novel target identification strategies in plant chemical biology. (A) Strategies relying on the affinity of the small molecule to isolate the target protein from a complex mixture such as a lysate or cellular environment. (B) The yeast-3-hybrid approach uses the activation of a transcriptional response by bringing together a DNA-binding domain and transcriptional activator via a fusion of the small molecule of interest and a known small molecule with high affinity for a known protein target. The latter is fused to the DNA-binding domain. The small molecule probes a cDNA library fused to the transcriptional activator. (C) Strategies relying on increased protein stability utilize small molecules to stabilize the increased dynamics, instability and degradation upon treatments such as denaturants or proteases, preventing or slowing down target protein degradation. Gray spheres: non-target proteins; blue spheres: target protein; orange cartoon: small molecule of interest; Lex A, Lex A DNA-binding domain; Mtx, methotrexate; DHFR, dihydrofolate reductase, target protein of Mtx; GAL4, GAL4 transcriptional activator.
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Figure 2: General principle of emerging and novel target identification strategies in plant chemical biology. (A) Strategies relying on the affinity of the small molecule to isolate the target protein from a complex mixture such as a lysate or cellular environment. (B) The yeast-3-hybrid approach uses the activation of a transcriptional response by bringing together a DNA-binding domain and transcriptional activator via a fusion of the small molecule of interest and a known small molecule with high affinity for a known protein target. The latter is fused to the DNA-binding domain. The small molecule probes a cDNA library fused to the transcriptional activator. (C) Strategies relying on increased protein stability utilize small molecules to stabilize the increased dynamics, instability and degradation upon treatments such as denaturants or proteases, preventing or slowing down target protein degradation. Gray spheres: non-target proteins; blue spheres: target protein; orange cartoon: small molecule of interest; Lex A, Lex A DNA-binding domain; Mtx, methotrexate; DHFR, dihydrofolate reductase, target protein of Mtx; GAL4, GAL4 transcriptional activator.

Mentions: The yeast 3-hybrid (Y3H) approach relies on the principles of the yeast 2-hybrid (Y2H) technology, but uses a modified small molecule of interest to allow interaction between the DNA-binding domain and the transcriptional activator (Figure 2). Initially (Licitra and Liu, 1996), Y3H was based on the Y2H system using the LexA DNA-binding domain and the trans-activation domain from the bacterial protein B42 (Gyuris et al., 1993). The so-called “hook” consisted of the LexA DNA-binding domain fused to the hormone-binding domain of the rat glucocorticoid receptor. The latter binds to dexamethasone, which is part of the hybrid small molecule comprising dexamethasone and FK506, or the “bait.” Finally, the “fish” consisted of human FKBP12 fused to the transcriptional activator B42. For screening purposes, FKBP12 represents any cDNA library of choice, whereas FK506 represents the small molecule of interest.


Target identification strategies in plant chemical biology.

Dejonghe W, Russinova E - Front Plant Sci (2014)

General principle of emerging and novel target identification strategies in plant chemical biology. (A) Strategies relying on the affinity of the small molecule to isolate the target protein from a complex mixture such as a lysate or cellular environment. (B) The yeast-3-hybrid approach uses the activation of a transcriptional response by bringing together a DNA-binding domain and transcriptional activator via a fusion of the small molecule of interest and a known small molecule with high affinity for a known protein target. The latter is fused to the DNA-binding domain. The small molecule probes a cDNA library fused to the transcriptional activator. (C) Strategies relying on increased protein stability utilize small molecules to stabilize the increased dynamics, instability and degradation upon treatments such as denaturants or proteases, preventing or slowing down target protein degradation. Gray spheres: non-target proteins; blue spheres: target protein; orange cartoon: small molecule of interest; Lex A, Lex A DNA-binding domain; Mtx, methotrexate; DHFR, dihydrofolate reductase, target protein of Mtx; GAL4, GAL4 transcriptional activator.
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Related In: Results  -  Collection

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Figure 2: General principle of emerging and novel target identification strategies in plant chemical biology. (A) Strategies relying on the affinity of the small molecule to isolate the target protein from a complex mixture such as a lysate or cellular environment. (B) The yeast-3-hybrid approach uses the activation of a transcriptional response by bringing together a DNA-binding domain and transcriptional activator via a fusion of the small molecule of interest and a known small molecule with high affinity for a known protein target. The latter is fused to the DNA-binding domain. The small molecule probes a cDNA library fused to the transcriptional activator. (C) Strategies relying on increased protein stability utilize small molecules to stabilize the increased dynamics, instability and degradation upon treatments such as denaturants or proteases, preventing or slowing down target protein degradation. Gray spheres: non-target proteins; blue spheres: target protein; orange cartoon: small molecule of interest; Lex A, Lex A DNA-binding domain; Mtx, methotrexate; DHFR, dihydrofolate reductase, target protein of Mtx; GAL4, GAL4 transcriptional activator.
Mentions: The yeast 3-hybrid (Y3H) approach relies on the principles of the yeast 2-hybrid (Y2H) technology, but uses a modified small molecule of interest to allow interaction between the DNA-binding domain and the transcriptional activator (Figure 2). Initially (Licitra and Liu, 1996), Y3H was based on the Y2H system using the LexA DNA-binding domain and the trans-activation domain from the bacterial protein B42 (Gyuris et al., 1993). The so-called “hook” consisted of the LexA DNA-binding domain fused to the hormone-binding domain of the rat glucocorticoid receptor. The latter binds to dexamethasone, which is part of the hybrid small molecule comprising dexamethasone and FK506, or the “bait.” Finally, the “fish” consisted of human FKBP12 fused to the transcriptional activator B42. For screening purposes, FKBP12 represents any cDNA library of choice, whereas FK506 represents the small molecule of interest.

Bottom Line: The current needs to understand gene function in plant biology increasingly require more dynamic and conditional approaches opposed to classic genetic strategies.Gene redundancy and lethality can substantially complicate research, which might be solved by applying a chemical genetics approach.Now understood as the study of small molecules and their effect on biological systems with subsequent target identification, chemical genetics is a fast developing field with a strong history in pharmaceutical research and drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Systems Biology, VIB Ghent, Belgium ; Department of Plant Biotechnology and Bioinformatics, Ghent University Ghent, Belgium.

ABSTRACT
The current needs to understand gene function in plant biology increasingly require more dynamic and conditional approaches opposed to classic genetic strategies. Gene redundancy and lethality can substantially complicate research, which might be solved by applying a chemical genetics approach. Now understood as the study of small molecules and their effect on biological systems with subsequent target identification, chemical genetics is a fast developing field with a strong history in pharmaceutical research and drug discovery. In plant biology however, chemical genetics is still largely in the starting blocks, with most studies relying on forward genetics and phenotypic analysis for target identification, whereas studies including direct target identification are limited. Here, we provide an overview of recent advances in chemical genetics in plant biology with a focus on target identification. Furthermore, we discuss different strategies for direct target identification and the possibilities and challenges for plant biology.

No MeSH data available.