Qunzhao Wang

Multicolor monitoring of dysregulated protein kinases in chronic myelogenous leukemia

The Bcr-Abl and Lyn protein tyrosine kinases have been separately linked to the emergence of imatinib resistance in patients with chronic myelogenous leukemia. We have developed fluorescent sensors for these kinases that are enzymatically and photophysically distinct, allowing us to simultaneously, yet separately, visualize the tyrosine kinase activities of both Abl and Lyn. Multicolor monitoring revealed that an imatinib-resistant cell line (MYL-R) displays a remarkable 13-fold enhancement in Lyn kinase activity relative to its imatinib-sensitive counterpart (MYL). By contrast, both cell lines display nearly identical Abl activities. The upregulation of Lyn kinase phosphotransferase activity in MYL-R cells is linked to an overexpression of the Lyn B isoform. Furthermore, MYL-R cells possess a 4-fold higher level of activated Lyn and 5-fold lower level of autoinhibited Lyn than MYL cells. Finally, studies with an activating SH2 ligand revealed that Lyn from imatinib-resistant MYL-R cells is primed and active, whereas Lyn from imatinib-sensitive cells is dependent upon phosphorylated SH2 ligands for activity.

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.

Monitoring of Protein Arginine Deiminase Activity by Using Fluorescence Quenching: Multicolor Visualization of Citrullination†

Epigenetics is driven by posttranslational modifications, such as methylation and acetylation of Arg and Lys residues. A less well publicized modification, namely conversion of Arg to citrulline (Cit), began to receive attention in the 1990s, when it was discovered that autoantibodies targeting citrulline-embedded epitopes are present in patients with rheumatoid arthritis.[1] The Arg-to-Cit conversion in histones has now been correlated with the regulation of gene expression.[2] PAD4, a member of the Protein Arginine Deiminase (PAD) enzyme family that catalyze this transformation, is overexpressed in rheumatoid arthritis, cancer, multiple sclerosis, and glaucoma.[3]Only a few assays have been described for the PAD enzyme family, including colorimetric[4], coupled[5], SDS-PAGE gel[6], affinity probe fluorescent polarization[7], and protein array[8] approaches. We describe herein a PAD4 activity fluorescence-based sensing strategy that can be employed throughout the visible spectrum and into the near infrared. The flexibility inherent within this strategy offers the ability to simultaneously monitor the activity of PAD4 along with other enzymes that modify histones. A particularly appealing biosensor design strategy employs fluorescently quenched substrates that experience relief from quenching upon conversion to product. For example, protease sensors have been designed that possess a fluorophore on one side of the scissile bond and a fluorescent quencher on the other.[9] Strategies developed for other enzymes employ fluorescent quencher molecules that are stripped away from the fluorophore-containing product.[10] It occurred to us that the PAD4-catalyzed conversion of a positively charged Arg residue (compound A) to a neutral Cit (compound C) should lead to the direct release of a noncovalently associated, negatively charged, quencher molecule (Q-) from a fluorescently quenched complex (compound B, Scheme 1). Furthermore, this strategy struck us as potentially applicable to a wide variety of fluorophores, thereby offering the flexibility to simultaneously visualize the action of PAD4 and other enzymes using wavelength-distinct sensors.[11] Scheme 1 The fluorescent PAD4 substrate A forms a non-fluorescent noncovalent complex B with a negatively charged quencher dye molecule. Upon citrullination of the PAD4 substrate, the now neutral product C loses its affinity for the dye, and a fluorescent response ... We prepared the following analogs of Ac-Ser-Gly-Arg-Gly-Ala, an efficient PAD4 substrate[12]: (a) a Gly-for-Ser substitution to preclude the possibility of phosphorylation when monitoring PAD4 activity in the presence of protein kinases (vide infra); (b) multimerized versions of (Gly-Arg-Gly-Ala)n to enhance the formation of the fluorescently quenched complex B (Scheme 1); (c) N-terminal attachment of the rhodamine derivative TAMRA and subsequently, upon identification of an optimized TAMRA-(Gly-Arg-Gly-Ala)nsubstrate, other fluorophores. A library of 47 commercially available dyes was screened against TAMRA-Gly-Arg-Gly-Ala and TAMRA-(Gly-Arg-Gly-Ala)3(2) to identify fluorescent quenchers (Supporting Information). Although TAMRA-Gly-Arg-Gly-Ala, with only a single Arg residue, is recalcitrant to significant fluorescent quenching, the fluorescence of the corresponding trimer is dramatically quenched by a variety of dyes. Several of the latter were examined for their ability to quench the fluorescence of TAMRA-(Gly-Arg-Gly-Ala)3 and generate a fluorescent enhancement upon exposure to PAD4 (Supporting Information). Our lead, Acid Green 27 (1), in combination with the peptide trimer, displays an impressive PAD4-catalyzed 57 ± 2-fold increase in fluorescence. Furthermore, the tetramer, TAMRA-(Gly-Arg-Gly-Ala)4, exhibits a 166 ± 10-fold increase in fluorescence and an analog, (Gly-Arg-Gly-Ala)2-Lys(TAMRA)-(Gly-Arg-Gly-Ala)2, displays a 138 ± 7-fold enhancement in fluorescence upon citrullination. The presumed resistance of the Cit-containing products(s) to Acid Green 27-mediated fluorescence quenching was confirmed by preparing authentic samples containing one, two, and three Cit residues. The fluorescence of Cit-containing analogs of TAMRA-GlyXaaGlyAlaGlyXaaGlyAlaGlyXaaGlyAla as a function of Acid Green 27 concentration is shown in Figure 1, where Xaa = Arg or Cit. Fluorescence quenching by Acid Green 27 is most pronounced as follows: (GRGA)3 > mono-substituted Cit > di-substituted Cit > tri-substituted Cit, supporting our hypothesis that the conversion of positively charged Arg to neutral Cit residues disrupts dye binding (Supporting Information). Figure 1 Fractional fluorescence of Cit-for-Arg TAMRA-(GRGA)3 derivatives as a function of Acid Green 27 concentration. Fractional fluorescence = fluorescence in the presence versus the absence of Acid Green 27. TAMRA-(GRGA)3= Arg••Arg••Arg ... Simultaneous visualization of the activity of multiple enzymes offers the possibility of concurrently monitoring the response of several biochemical pathways to environmental stimuli. However, the response of the sensor associated with each enzymatic activity must be wavelength distinct. This lead us to explore the range of fluorophores that respond to the Scheme 1 strategy, which offers an assessment of the flexibility of PAD4 assay to work in concert with reporters of other enzyme-catalyzed reactions. We prepared Fl-(GRGA)3 derivatives where Fl = DEAC (3; λem 480 nm), Oregon Green 488 (4; λem 525 nm), Cascade Yellow (5; λem 545 nm), Rox (6; λem 591 nm), Texas Red (7; λem 615 nm), and Atto 655 (8; λem 680 nm). Each Fl-(GRGA)3 derivative was screened with the quencher library (Supporting Information). With the exception of Oregon Green 488, all substrates are strongly susceptible to quenching, most notably by Acid Green 27: DEAC (49 ± 1-fold @ 480 nm) Cascade Yellow (55 ± 2-fold @ 545 nm), Rox (133 ± 9-fold @ 610 nm), Texas Red (156 ± 10-fold @ 610 nm), and Atto 655 (48 ± 5-fold @ 680 nm). The large fluorescence changes associated with the PAD4 substrates are easily visualized without sophiscated instrumentation. The DEAC, Cascade Yellow, TAMRA and Atto 655 derivatives have very different emision wavelengths and good conversion rates, and thus were selected for side-by-side photoimaging studies. With the exception of DEAC-(GRGA)3, for which a Xe flash lamp and proper filters were employed to eliminate UV light interference with the camera, fluorophore excitation was performed with a hand-held UV lamp and emission was photographed with a Vis-IR camera (Figure 2). In an analogous fashion, we captured color images directly from the spectrofluorimeter by integrating the Vis-IR camera with the sample chamber (Supporting Information). Figure 2 Visualization of Fluorophore-(GRGA)3 PAD4 substrates. The DEAC-peptide was excited with a Xe Flash lamp and all others with a Hg arc lamp. Each sample set was incubated with (left) and without (right) PAD4 for 60 min prior to image capture. The diversity of fluorophores compatible with PAD4 catalysis furnishes the flexibility to assemble paired fluorescent enzyme sensors that enable simultaneous monitoring of multiple enzymatic activities. For example, histones are known to suffer a wide variety of enzyme-catalyzed modifications which, in turn, influence gene expression. The cAMP-dependent protein kinase (PKA) is the first enzyme shown to phosphorylate histones.[13] We paired the PAD4 TAMRA-(GRGA)3 sensor with a previously described PKA peptide-based sensor, Ac-GRTGRRDap(pyrene)SYP-amide, that reports phosphorylation via a pyrene fluorophore.[5, 6] The fluorescence changes of PAD4 and PKA substrates were monitored at 590 and 400 nm, respectively. An increase in PKA substrate fluorescence is only observed in the presence of PKA and, similarly, an increase in PAD4 substrate fluorescence is only observed in the presence of PAD4 (Figure 3). The activity of both enzymes can be simultaneously monitored and the observed activity under combined conditions for each enzyme is the same as that of the enzymes alone. Finally, we examined the ability of the molecular construct 3 to detect endogenous PAD4 activity in lysates from a human promyelocytic leukemia cell line (HL-60). Since PAD4 is only found in the nucleus (confirmed by Western blot analysis; Figure 4b), we compared PAD4 activities in nuclear and cytoplasmic extracts (Figure 4a). As expected, only the nuclear extract displays PAD4 activity. Furthermore, the observed deiminase activity is Ca2+-dependent and blocked by the PAD4 inhibitor minocycline, which is both consistent with and reflective of PAD4 action on sensor 3. Figure 3 Multicolor monitoring of PKA and PAD4 activity. a) Observed change in relative fluorescence units (λex 340 nm; λem 400 nm) of the PKA substrate in the presence of PAD4 (red), PKA (green), and both enzymes (blue). b) Observed change in ... Figure 4 (a) Fluorescence change of PAD4 substrate 3/quencher 2 pair upon exposure to HL-60 nuclear extract in the presence of Ca2+ (black); HL-60 cytoplasmic extract in the presence of Ca2+(yellow); HL-60 nuclear extract in the absence of Ca2+(green); HL-60 nuclear ... In summary, we have created a series of PAD4 sensors that furnish a robust fluorescent response across the visible spectrum and into the near infrared. Given the demonstrated biological and biomedical significance of multiplexing nucleic acid and protein content from cells, sensors that are tunable to specific windows throughout the visible and near IR potentially enable the simultaneous monitoring of multiple enzymatic activities.

The Plasma Membrane as a Reservoir, Protective Shield, and Light-Triggered Launch Pad for Peptide Therapeutics

Although peptide-based therapeutics are finding increasing application in the clinic, extensive structural modification is typically required to prevent their rapid degradation by proteases in the blood. We have evaluated the ability of erythrocytes to serve as reservoirs, protective shields (against proteases), and light-triggered launch pads for peptides. We designed lipidated peptides that are anchored to the surface of red blood cells, which furnishes a protease-resistant environment. A photocleavable moiety is inserted between the lipid anchor and the peptide backbone, thereby enabling light-triggered peptide release from erythrocytes. We have shown that a cell-permeable peptide, a hormone (melanocyte stimulating hormone), and a blood-clotting agent can be anchored to erythrocytes, protected from proteases, and photolytically released to create the desired biological effect.

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells.

Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system that utilizes a light-programmable, cell-permeable reporter deliverable simultaneously to many cells. The reporters ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2-4,5-dimethoxy 2-nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single-cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity.

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.

Multicolor monitoring of the proteasome's catalytic signature.

The proteasome, a validated anticancer target, participates in an array of biochemical activities, which range from the proteolysis of defective proteins to antigen presentation. We report the preparation of biochemically and photophysically distinct green, red, and far-red real-time sensors designed to simultaneously monitor the proteasome’s chymotrypsin-, trypsin-, and caspase-like activities, respectively. These sensors were employed to assess the effect of simultaneous multiple active site catalysis on the kinetic properties of the individual subunits. Furthermore, we have found that the catalytic signature of the proteasome varies depending on the source, cell type, and disease state. Trypsin-like activity is more pronounced in yeast than in mammals, whereas chymotrypsin-like activity is the only activity detectable in B-cells (unlike other mammalian cells). Furthermore, chymotrypsin-like activity is more prominent in transformed B cells relative to their counterparts from healthy donors.