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Metabolism of peptide reporters in cell lysates and single cellsThis article is part of a themed issue highlighting the targeted study of single units, such as molecules, cells, organelles and pores - The "Single" Issue, guest edited by Henry White.Electronic supplementary information (ESI) available. See DOI: 10.1039/c2an16162a

The stability of an Abl kinase substrate peptide in a cytosolic lysate and in single cells was characterized. In the cytosolic lysate, the starting peptide was metabolized at an average initial rate of 1.7 ± 0.3 zmol pg −1 s −1 with a t 1/2 of 1.3 min. Five different fragments formed over time; howe...

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Bibliographic Details
Main Authors: Proctor, Angela, Wang, Qunzhao, Lawrence, David S, Allbritton, Nancy L
Format: Article
Language:English
Online Access:Get full text
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Summary:The stability of an Abl kinase substrate peptide in a cytosolic lysate and in single cells was characterized. In the cytosolic lysate, the starting peptide was metabolized at an average initial rate of 1.7 ± 0.3 zmol pg −1 s −1 with a t 1/2 of 1.3 min. Five different fragments formed over time; however, a dominant cleavage site was identified. Multiple rational design cycles were utilized to develop a lead peptide with a phenylalanine and alanine replaced by an ( N -methyl)phenylalanine and isoleucine, respectively, to attain cytosolic peptidase resistance while maintaining Abl substrate efficacy. This lead peptide possessed a 15-fold greater lifetime in the cytosolic lysate while attaining a 7-fold improvement in k cat as an Abl kinase substrate compared to the starting peptide. However, when loaded into single cells, the starting peptide and lead peptide possessed nearly identical degradation rates and an altered pattern of fragmentation relative to that in cell lysates. Preferential accumulation of a fragment with cleavage at an Ala-Ala bond in single cells suggested that dissimilar peptidases act on the peptides in the lysate versus single cells. A design strategy for peptide stabilization, analogous to that demonstrated for the lysate, should be effective for stabilization in single cells. Peptide cleavage sites occurring during incubation in lysates and single cells were identified and non-native amino acids inserted to stabilize the peptide. Multiple iterative cycles yielded a peptidase-resistant, kinase-substrate peptide.
ISSN:0003-2654
1364-5528
DOI:10.1039/c2an16162a