Angela Proctor

Metabolism of peptide reporters in cell lysates and single cells

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.

Measurement of Protein Kinase B Activity in Single Primary Human Pancreatic Cancer Cells

An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg–1 s–1, for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.

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

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 widespread utility in both lysate-based and single-cell assays of kinase activity.

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells

Background & Aims Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished. Methods The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. Results The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. Conclusions This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.

β-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment

A strategy was developed to extend the lifetime of an peptide-based substrate for Abl kinase in the cytosolic environment. Small β-turn structures were added to the peptides N-terminus to block entry into peptidase catalytic sites. The influence of the size of the β-turn and two covalent cross-linking strategies on the rate of hydrolysis was assessed. The most peptidase-resistant substrate was degraded at a rate of 0.6 pmol mg(-1) s(-1) and possessed a half-life of 20.3 ± 1.7 min in a Baf/BCR-ABL cytosolic lysate, representing 16- and 40-fold improvements, respectively, over that of a control peptide lacking the β-turn structure. Furthermore, the kcat/KM value of this peptide was 432 μM(-1) min(-1), a 1.25× increase over the unmodified control, verifying that the added β-turn did not hinder the substrate properties of the peptide. This improved peptide was microinjected into single Baf/BCR-ABL cells and substrate phosphorylation measured. Zero to forty percent of the peptide was phosphorylated in the single cells. In contrast, when the control peptide without a β-turn was loaded into cells, the peptide was too rapidly degraded to detect phosphorylation. This work demonstrates that small β-turn structures can render peptides more resistant to hydrolysis while retaining substrate efficacy and shows that these stabilized peptides have the potential to be of high utility in single-cell enzyme assays.

Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity

Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology in vitro, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Nonproteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both in vitro and in cellulo. The reporter contains a conformationally constrained phosphorylatable moiety (7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of L-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter.

Fluorous enzymatic synthesis of phosphatidylinositides

A fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficiently synthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorous polytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions.

Chemical fixation to arrest phospholipid signaling for chemical cytometry

Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by single-cell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followed by glutaraldehyde fixation and immediate analysis, 24±2% and 139±12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24h yielded recoverable lipid of 61±3% (C6-PIP2) and 55±5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to single-cell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.

Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes

The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.

Abstract 3345: Measurement of Akt activity in single primary human pancreatic cancer cells using chemical cytometry

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Genetic alterations in signaling pathways and processes, including mutations, deletions, and amplifications, have been found in many pancreatic cancers. Prominent among these pathways is the phosphoinositide 3-kinase (PI3-K) pathway, which regulates multiple functions, including: proliferation; stress response; and apoptosis. Akt (also known as protein kinase B or PKB) is a serine/threonine kinase in the PI3-K pathway whose activity has been linked to providing cancer cells with anti-apoptotic properties. In pancreatic cancer, constitutive PI3-K/Akt activity appears to be an indicator of tumor aggressiveness, with high levels of active Akt associated with decreased patient survival. Currently, the gold standard for assessment of Akt activity is Western blot, where cellular homogenates are probed with anti-Akt antibodies to measure active Akt. However, this technology lacks single-cell resolution and yields an average result for a population of cells. Heterogeneity of Akt activity in single cells is thought to be an important mechanism in chemotherapy resistance, and analysis of multiple cell types suggests that Akt activity may be bimodal. A population average-based measurement would fail to capture the existence of high and low states of activity, whereas single-cell interrogation should reveal this behavior. We describe the single-cell measurement of Akt activity in single human pancreatic cancer cells. An optimized peptide substrate incorporating non-native residues for peptidase resistance was used to measure Akt activity within cells. Briefly, the peptide was microinjected into a single cell, where it was available for phosphorylation by Akt. Single cells were lysed and their contents loaded into an overlying fused-silica capillary for electrophoretic separation of the non-phosphorylated and phosphorylated peptide. We first validated the system in 3 model pancreatic cancer cell lines before applying it to primary cells from human pancreatic cancer xenografts. Some tumor cell lines exhibited statistically significant bimodal behavior in the level of Akt activity. In the primary cells, Akt exhibited highly variable activity at the single-cell level, with some cells showing little to no activity while others demonstrated very high levels of activity as indicated by nearly complete phosphorylation of the substrate peptide. Single-cell Akt activity was blocked in the presence of wortmannin, a PI3-K inhibitor. This system also enabled characterization of peptidase action on the substrate with primary cells demonstrating a 10-fold lower level of peptidase activity. Future work focuses on utilizing this technology for higher-throughput analysis of Akt activity in primary patient tissue from multiple cancer types. Citation Format: Angela Proctor, S. Gabriela Herrera-Loeza, Jen Jen Yeh, Nancy L. Allbritton. Measurement of Akt activity in single primary human pancreatic cancer cells using chemical cytometry. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3345. doi:10.1158/1538-7445.AM2014-3345