Issue 21, 2016

Development of a protease-resistant reporter to quantify BCR–ABL activity in intact cells

Abstract

A peptidase-resistant ABL kinase substrate was developed by identifying protease-susceptible bonds on an ABL substrate peptide and replacing flanking amino acids with non-native amino acids. After an iterative design process, the lead, or designed, peptide X-A possesses a six-fold longer life in a cytosolic lysate than that of the starting peptide. The catalytic efficiency (kcat/KM) of purified ABL kinase for the lead peptide (125 s−1 μM−1) is similar to that of the starting peptide (103 s−1 μM−1) demonstrating preservation of the peptide's ability to serve as a kinase substrate. When incubated in cytosolic lysates, the lead peptide is slowly degraded into 4 fragments over time. In contrast, when loaded into intact cells, the peptide is metabolized into 5 fragments, with only 2 of the fragments corresponding to those in the lysate. Thus the two environments possess differing peptidase activities, which must be accounted for when designing peptidase-resistant peptides. In both settings, the substrate is phosphorylated by BCR–ABL providing a readout of BCR–ABL activity. A small panel of tyrosine kinase inhibitors verified the substrate's specificity for BCR–ABL/ABL kinase activity in both lysates and cells in spite of the multitude of other kinases present. The designed peptide X-A acts as a long-lived BCR–ABL kinase reporter in the leukemic cells possessing the BCR–ABL mutation.

Graphical abstract: Development of a protease-resistant reporter to quantify BCR–ABL activity in intact cells

Supplementary files

Article information

Article type
Paper
Submitted
17 Jūn. 2016
Accepted
07 Sept. 2016
First published
07 Sept. 2016

Analyst, 2016,141, 6008-6017

Author version available

Development of a protease-resistant reporter to quantify BCR–ABL activity in intact cells

A. Proctor, I. G. Zigoneanu, Q. Wang, C. E. Sims, D. S. Lawrence and N. L. Allbritton, Analyst, 2016, 141, 6008 DOI: 10.1039/C6AN01378C

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