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 Чер 2016
Accepted
07 Вер 2016
First published
07 Вер 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

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements