Metabolism of peptide reporters in cell lysates and single cells

Abstract

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 t1/2 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 kcat 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.

Development of β-Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

“…To test this, the OWRWR peptide was incubated with OPM2 lysates at 37 °C to mimic the intracellular environment of multiple myeloma cells. After varying the incubation times, peptide fragmentation was assayed by separating the mixture using CE and identifying peptide fragments from their fluorescence and migration times as previously described by Proctor et al 18 The OWRWR peptide was resistant to the degradation in the OPM2 lysates (Figure 4, black squares), with 80% of the full-length peptide remaining after 180 min, yielding a half-life of 630 min for the intact peptide in the cytosolic mixture. Conversely, the scrambled version of the OWRWR peptide (termed Scram-OWRWR, sequence in Figure 1A) was almost completely degraded in the OPM2 lysates within 60 min (Figure 4, blue circles), with a half-life of 30 min.…”

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

“…30 While V-48B exhibits a 15-fold improved half-life in the face of cellular proteases relative to its parent sequence III-67B (Table 1), V-48B’s degradation resistance is insufficient for kinase activity measurement when faced with the high concentration of peptidases within an intact cell. This is primarily due to hydrolysis of the peptide bond between the two alanine residues.…”

“…The supernatant was collected after centrifugation and total protein concentration was determined using fluorescamine. 30 For assays, cytosolic lysate (3 mg mL −1 total protein) was incubated with 1 μM peptide substrate at 37 °C. Aliquots were collected at different time points and the reaction inactivated with twice the aliquot volume of 200 mM HCl.…”

“…30 a t 1/2 is the time at which 50% of the peptide was degraded in the cytosolic lysate (3 mg mL −1 total protein). b t 50% P is the time required for 50% of the peptide to be phosphorylated by Abl-1 kinase under the conditions employed using 12 nM of kinase. NP indicates no phosphorylation occurred.…”

“…30 Our design strategy utilizes non-native amino acids incorporated into the reporter at specific loci to impart stability to a peptide substrate that is otherwise rapidly degraded by intracellular proteases. Non-native amino acids are often used to expand the chemical properties available for proteins and peptides by selectively altering individual residues to feature properties not attainable with native amino acids.…”