Quality control of fluorescence polarization assay over gel-based assay.

Abstract

Desmoplakin (DSP) is an integral part of the desmosome, the intercellular junction that links the intermediate filaments of adjacent cells. Specific mutations in DSP (R451G, S507F, S442F, and S299R) result in calpain hypersensitivity. Our long-term objective is to reduce this hypersensitivity by occluding the protease binding site of DSP with small molecule drugs or other molecules. To do this type of screening efficiently, we need a reliable, robust, and inexpensive DSP screening method. However, existing DSP expression constructs are low yield. Also, existing methods of observing degradation rates are done via a protein and time-intensive gel-based assay. Last, calpain is expensive. Here we describe a new fluorescence polarization assay to detect DSP degradation using both a shorter fluorescein isothiocyanate (FITC) labeled DSP construct and using trypsin as the protease. We show that this shorter construct is susceptible to degradation however the mutants degrade at a faster rate. We also show that trypsin was a reasonable substitute for calpain. Having now developed these new reagents and methods, we are well-positioned to screen for ways to rescue DSP mutants.