With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.
- MeSH
- Arabidopsis genetika MeSH
- cytosol imunologie metabolismus MeSH
- fosfotransferasy biosyntéza genetika imunologie MeSH
- mapy interakcí proteinů genetika imunologie MeSH
- proteinkinasy biosyntéza genetika imunologie MeSH
- proteiny huseníčku biosyntéza genetika imunologie MeSH
- protilátky aplikace a dávkování imunologie MeSH
- regulace genové exprese u rostlin MeSH
- rekombinantní proteiny aplikace a dávkování imunologie MeSH
- signální transdukce MeSH
- umlčování genů imunologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²⁺, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF₃⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²⁺. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.
- MeSH
- Arabidopsis enzymologie genetika fyziologie MeSH
- fosforylace MeSH
- fosfotransferasy genetika metabolismus MeSH
- histidin metabolismus MeSH
- krystalografie rentgenová MeSH
- mapování interakce mezi proteiny MeSH
- molekulární modely MeSH
- mutace MeSH
- proteinkinasy chemie genetika izolace a purifikace metabolismus MeSH
- proteiny huseníčku chemie genetika izolace a purifikace metabolismus MeSH
- rekombinantní fúzní proteiny MeSH
- senzitivita a specificita MeSH
- signální transdukce fyziologie MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Repair of damaged DNA is a dynamic process that requires careful orchestration of a multitude of enzymes, adaptor proteins, and chromatin constituents. In this issue of Cell, Lisby et al. (2004) provide a visual glimpse into how the diverse signaling and repair machines are organized in space and time around the deadliest genetic lesions--the DNA double-strand breaks.
- MeSH
- buněčné jádro * genetika metabolismus ultrastruktura MeSH
- diagnostické zobrazování metody přístrojové vybavení MeSH
- DNA * genetika metabolismus MeSH
- fosfotransferasy genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- lidé MeSH
- oprava DNA * genetika MeSH
- poškození DNA * genetika MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- MeSH
- alely MeSH
- bipolární porucha genetika komplikace MeSH
- finanční podpora výzkumu jako téma MeSH
- fosfotransferasy genetika MeSH
- genetická variace MeSH
- lidé MeSH
- mozek metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- schizofrenie genetika komplikace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- dítě MeSH
- fosfotransferasy genetika nedostatek MeSH
- hypergamaglobulinemie enzymologie genetika MeSH
- kyselina mevalonová metabolismus MeSH
- lidé MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
