Scientific journal
Scientific Review. Biological science
ISSN 2500-3399
ПИ №ФС77-57454

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FRET-SENSOR FOR DETERMINATION OF CASPASE-8 ACTIVITY

Zherdeva V.V. 1 Ivashina T.V. 1, 2 Goryashchenko A.S. 1 Savitsky A.P. 1
1 A.N. Bach Institute of biochemistry RAS
2 G.K. Skryabin Institute of biochemistry and physiology of microorganisms RAS»
Apoptosis is a general biological mechanism which is responsible for maintaining constant the number of cell populations and culling of defective cells. Dysregulation of apoptosis leads to the emergence of various diseases associated with increased or, conversely, inhibition of apoptosis. Specific cysteine proteases - caspases - are responsible for the morphological and biochemical changes that occur during apoptosis. The main effector enzyme in apoptosis is caspase-3, whereas the earlier event marker of apoptosis is the caspase-8 activation. Simultaneous detection of the activity of caspases 3 and 8 will help to understand whether running process of apoptosis or activation of caspase-8 is caused by different scenario of the cellular response, at what stage is the cell apoptosis and activation pathway of apoptosis. The aim of this study was to characterize properties of genetically encoded caspase-8 FRET-sensor TagRFP-IETD-KFP which is based on red fluorescent proteins TagRFP and KFP. The dynamic light scattering experiments showed that hydrodynamic radius of the sensor was 5.9 nm, which in spherical model approach corresponds to the molecular weight of 213 kDa and tetrameric form of the protein in solution. FRET efficiency value in the sensor was calculated from the spectral data and was 56%. Finally, by means of fluorescence intensity and fluorescence lifetime measurements efficient hydrolysis of the sensor by caspase-8 in vitro was demonstrated.