Development of Ratiometric Fluorescence Sensing Molecule for Caspase-14, a Key Enzyme of Epidermis Metabolism

Abstract

Skin is the vastest organ harboring principle functions to protect from external stimuli and internal moisture desorption. The loss of functions might bring about diseases and senescence and well maintained metabolisms are recognized as crucial points to prevent those situations. But due to complexities of skin tissues, there would be some difficulties in progress of elucidation for skin metabolisms in detail. Various differentiated tissues layered in order can form dermis and epidermis to exfoliate as scurf in the top layer finally. Caspase-14 categorized in cysteine-aspartic acid protease (Caspase) family that has been identified as key enzymes to promote programmed cell deaths by sequential activations is known to be specifically expressed in the upper tissues and involved in the differentiation, but not verified to have relationship with final exfoliation processes at the time of cell death. We then developed Fluorescence Resonance Energy Transfer (FRET) based sensing molecules to monitor Caspase-14 activity quantitatively. We utilized Green Fluorescent Protein (GFP) and organic fluorescent dyes because FRET phenomena could be achieved under condition with regulated interactions between at least two fluorescent molecules. The elaborately designed fluorescent complexes could render quantifiable sensing over fluorescent background but be simply introduced into cells via endocytic mechanisms to detect intracellular enzymatic activities alive. In this study, we demonstrated Caspase-14 activation during apoptosis induction in cultured normal epidermal cells that suggested possibilities of Caspase-14 contribution to programmed cell deaths.