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Characterisation of a caspase-like protein activated by the SI response in Papaver rhoeas

Reference	BB/D521181/1
Principal Investigator / Supervisor	Professor Vernonica E. (Noni) Franklin-Tong
Co-Investigators / Co-Supervisors
Institution	University of Birmingham
Department	Sch of Biosciences
Funding type	Research
Value (£)	290,477
Status	Completed
Type	Research Grant
Start date	17/10/2005
End date	16/07/2009
Duration	45 months

Abstract

There are 3 major aims for this project: (1) Purification and identification of the DEVDase activated by SI. (2) Identification of cellular targets of the caspase-like activity. (3) Identification of cellular targets of the caspase-like activity. Approaches will include: (1) Purification and identification of the DEVDase activated by SI. In order to achieve this objective we propose to use an affinity purification approach to purify and identify the DEVDase protein activated by SI in poppy pollen. The tagged probe, DCG04-biotin, reacts and binds to activated cysteine proteases, thus allowing enrichment of fractions with the caspase-3-like (DEVDase) activity. Crude pollen protein extracts will be labelled with DGC-04 and incubation with magnetic streptavidin beads will allow elution of bound proteins which can be analysed by SDS-PAGE or 2D gels. Alternatively, biotinylated DEVD could be used in a similar manner, as we know this specifically inhibits the caspase-like activity. Comparisons of the protein extracted from untreated pollen and SI-stimulated pollen will enable identification of activated DEVDases. Proteomic analysis of the enriched fractions should aid identification of specific proteins, which can be excised and analysed using QTOF mass spectrometry. This will allow us to obtain amino acid sequence and thus provide sufficient information to clone the poppy DEVDase/caspase-3-like gene using RT-PCR using suitably designed primers. We have a poppy pollen cDNA library already constructed. (2) Characterisation of the caspase-like activity triggered during SI in poppy pollen. We propose to use a range of synthetic inhibitor peptides (e.g. Ac-DEVD-CHO) together with more general protease inhibitors to enable detailed characterisation of the caspase-like activity triggered by SI. Many caspase protease activity assay kits are commercially available. The AFC-based tetrapeptide substrates (e.g. Ac-DEVD-AFC) give a change in fluorescence upon protease cleavage, so actas indicators of caspase activities, which can be measured. This approach will be used to further establish the activity of the SI-simulated caspase-like protein. Pollen samples with SI-induced PCD will be compared with untreated pollen. Pollen extracts will be incubated with the AFC-conjugated caspase substrate and increases in a specific caspase activity can be measured. Other types of caspase assay kits will allow us to measure caspase activities in living pollen. The labelled caspase inhibitor peptide (e.g. FAM-DEVD-FMK) enters living cells and irreversibly binds to the caspase active site, and so real-time activity (fluorescence) can be imaged or quantified. (3) Identification of cellular targets of the caspase-like activity will utilise a proteomics approach. Samples from SI-challenged pollen (compared with untreated) at various time points after SI induction will be analysed on 2D gels to identify protein modification and potential cleavage products. This will allow identification of target proteins and establish detailed temporal events. QTOF or FT-ICR mass spectroscopy will allow analysis of the proteins identified.

Summary

Programmed Cell Death (PCD) is an extremely important and widespread process. It involves the highly regulated death of targeted cells during development, where it plays a role in ensuring that organs are correctly sculpted. For example, fingers and toes originate as webbed digits and during development signals tell particular cells to die, thereby causing separation of the fingers. In plants, the holes in Swiss cheese plants are formed in a similar manner. PCD is also used in defence against pathogens in both animals and plants as a result of signals received from the pathogen. My lab studies self-incompatibility (SI), which is an important mechanism used by plants to prevent self-fertilisation, which would lead to inbreeding, which is not desirable. The female part of the flower (stigma) secretes a protein that selectively inhibits incompatible (self) pollen, but not cross pollen. We recently showed that PCD is triggered in incompatible pollen and is used to inhibit self pollen tube growth. A specific protein from the stigma triggers a signal in the incompatible pollen grain that starts a cascade of events that tell it to die. A key protein involved in apoptosis and PCD in animal cells is caspase-3. This is a protease, so has an activity that cleaves proteins. This caspase acts as an executioner, and when activated, it cleaves many cellular proteins which eventually result in cell death. Cleavage of proteins is not random, but highly specific, and caspase-3 has to find a string of four particular amino acids in the correct order (DEVD) in order to be able to cut the protein up at that site. Specific drugs have been designed as inhibitors for this interaction; they act by mimicking this 4 letter region and so interfere with caspase recognition. As there are many caspases (all with different 4 letter code specificities), these inhibitors are useful to identify proteins with a specific caspase activity. As mentioned earlier, in animal cells the caspase-3 activity with DEVD specificity is the key protein involved in PCD. The gene encoding this protein has been cloned and sequenced. Since the Arabidopsis genome sequence is available, it is possible to look for the gene corresponding the caspase-3 in plants. However, no similar sequence has been found. So, although there is good evidence that plants have caspase-3 like activity (with DEVD specificity, just like in animal cells), the gene must be different. Obviously identification of the equivalent gene from plants is of great importance, because it plays such a critical role in regulation of life or death. This project aims to clone the caspase-3-like gene from poppy pollen. The protein will be purified using drugs with the specific 4 letter code specified to caspase-3 to pull it out. We can then obtain the amino acid sequence of the protein, which will allow us to clone the gene encoding it. Another aim is to characterise the enzyme activity of this protein, using drugs which are labelled fluorescently so we can detect activity. Finally, we hope to identify the proteins that are the cellular targets of the caspase-3-like activity, and that are cleaved.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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