Design of genetically encded probes to detect peptidase activity in vivo.

PEREZ, BRIAN; NIEMIROWICZ, GABRIELA T.; CAZZULO, JUAN JOSÉ; ALVAREZ, VANINA EDER; BOUVIER, LEÓN ALBERTO

Buenos Aires

Congreso; Reunión conjunta de las Sociedades de Biociencias; 2017

Proteases play a major role in regulating a wide range of physiological and pathological processes. Unregulated protease activation or inhibition could eventually result in a number of important diseases including cancer, atherosclerosis, and neurodegenerative diseases.Currently, protease activity can be determined by several methods. Western blot analysis and quantitative real-time polymerase chain reaction can estimate the expression levels of the enzymes and their inhibitors although limited information for understanding the dynamic activity of the proteases in vivo is provided. One of the most commonly applied methods is the activity measurement in cell lysates using small chromogenic or fluorogenic synthetic peptides that are, in many cases, commercially available. Although some probes are able to penetrate cells they cannot be delivered into specific sub-cellular compartments. Moreover, they are not ideal for continuous measurements due to their limited lifetime, specificity and stability.In this work we report a strategy to create protease sensors by grafting an enzymatic cleavage linker into a semsitive location for changing chromophore properties of enhanced green fluorescent protein (EGFP) following protease cleavage. We first validated the system in Escherichia coli by expressing and purifying the protease and FRET sensors, using the TEV protease as model enzyme.Once the cleavage was confirmed by Western blot analysis and by changes in the emission spectra, we moved to in vivo studies in Saccharomyces cerevisiae were the same analyses were performed.This strategy opens a new avenue for developing specific protease sensors to investigate enzymatic activity in real time, to probe enzymatic relevance corresponding to proteases in vitro and in vivo, and to screen protease inhibitors with therapeutic effects.