Development of a Peptidase-Resistant Substrate for Single-Cell Measurement of Protein Kinase B Activation

Abstract

An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg−1 s−1 and 0.04 ± 0.08 zmol pg−1 s−1, respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find wide-spread utility in both lysate-based and single-cell assays of kinase activity.

N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides

“…The N-gemini peptides 4 – 6 , in contrast, exhibit a 24-, 28-, and 7.5-fold increase in protease resistance of the peptide dimers over their un-dimerized counterparts, despite the fact that they are still unstructured (Figure S4 and Table 1). This increase in protease resistance is on the same order as the best methods reported to date, including appending a cyclic β-hairpin, 14 which is synthetically more challenging, or incorporation of unnatural amino acids within the substrate itself, 16,17 which requires significantly more optimization of both degradation resistance and substrate activity. The increased resistance is presumably due to the fact that the peptide dimers can only enter the catalytic cleft of peptidases starting with the C-terminus, hindering their recognition by the peptidases.…”

“…We demonstrate that N-terminal dimerization improves the half-lives of unstructured peptides by up to 28-fold, which is on par with protection of an unstructured peptide by capping with N-terminal cyclic β-turn 14,15 or by incorporation and optimization of unnatural amino acids into the substrate sequence. 16,30 Importantly, the approach reported here is far more synthetically accessible, thus providing a significant advancement over previous methods and a new tool for achieving protease-resistant unstructured peptides.…”

“…14,15 A common approach involves incorporation of unnatural amino acids into the sequence. 16,17 However, this approach often requires an iterative design process that can be time consuming and costly, as a delicate balance between resistance and peptide efficacy must be maintained. Furthermore, while a wide range of peptide stapling methodologies, 18,19 hydrogen bond surrogates, 20,21 and mixed α/β-peptides 22–24 have been developed, these are limited to α-helical peptides.…”

“…14,16 However, peptide degradation is competitive with phosphorylation, 14,16,28 making such an assay impossible without some method for increasing protease resistance (Scheme 1). …”

Identification of a p53-based portable degron based on the MDM2-p53 binding region

“…6 Peptide reporters can be rapidly synthesized, easily modified, and do not require complex manipulations for incorporation into single, living cells. Previous work utilized peptide-based reporters to monitor peptidase, kinase 7 and phosphatase 8 activity in single cancer cells. One potential drawback with peptide-based reporters is the rapid degradation by intracellular peptidases and proteases.…”