Tatsuhiko Sonoda

An intracellular kinase signal-responsive gene carrier for disordered cell-specific gene therapy

We have previously reported artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) using cationic polymer. This cationic polymer (PAK) was a graft-type polymer with an oligopeptide that is a substrate for PKA and could regulate gene-expression in a cell-free system. In the present study, we carried out a detailed characterization of the PAK-DNA complex (AFM observation and DLS measurement) and tried to apply this polymer to living cells. In the unstimulated NIH 3T3 cells, transfection of the PAK-DNA complex showed no expression of the delivered gene. This means that PAK formed a stable complex with DNA in the normal cells to totally suppress gene expression. In contrast, significant expression was seen when the PAK-DNA complex was delivered to forskolin-treated cells. Thus, activated PKA disintegrates the complexes even in living cells, resulting in gene expression. Our results indicate that this type of intracellular signal-responsive polymer will be useful for the cell-specific release of genes.

Optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis.

We investigated the optimal surface chemistry of peptide immobilization for on-chip phosphorylation analysis. In our previous study, we used a heterobifunctional cross-linker sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxalate (SSMCC) to immobilize cysteine-terminated peptides on an amine-modified gold surface. The study revealed that the phosphorylation efficiency and rate were low (only 20% at 2 h) comparing with the reaction in solution. In this study, to improve the phosphorylation efficiency, the kinase substrates were immobilized via poly(ethylene glycol) (PEG), a flexible, hydrophilic polymer. An improvement in cSrc phosphorylation was achieved (60% at 1 h) from using a PEG-inserted peptide and SSMCC. However, no phosphorylation could be detected when the peptide was immobilized with a PEG-containing cross-linker. Fluorescence-labeled peptide studies revealed that the use of longer cross-linkers resulted in lower immobilization density. We considered that the flexible PEG linker was preferable to secure high phosphorylation efficiency for the immobilized peptide, probably due to the improvement of cSrc accessibility and peptide mobility, but the immobilization protocol is critical for keeping high density of the peptide immobilization. In addition, such an accelerating effect of PEG linker against on-chip phosphorylation of an immobilized peptide may depend on kinase structures or the position of the active center, because no improvement of on-chip peptide phosphorylation was observed in protein kinase A. However, PEG linker also did not suppress the phosphorylation in protein kinase A. Thus, we concluded that SSMCC and PEGylated peptide will be a good combination for the surface chemistry of on-chip phosphorylation in peptide array.

Surface plasmon resonance imaging measurements of caspase reactions on peptide microarrays

Enzymatic activity monitoring of caspases, which are a class of cysteine protease, was performed by using peptide arrays based on surface plasmon resonance (SPR) imaging. The strategy of the detection is straightforward, using streptavidin to amplify the SPR signals of the surface-immobilized substrate peptides labeled with biotin at the C termini. Thus, the cleavage of the substrate peptides by caspases was detected as a signal decrease. Using this method, we succeeded in monitoring the activities of purified caspases and caspases in cell lysates. The SPR imaging-based peptide array would be applicable to cell-based drug screening and biochemical studies to reveal signal transduction processes.

Protein Kinase Substrate Profiling with a High-Density Peptide Microarray

We describe a powerful peptide microarray for profiling protein kinase substrates that combines the merits of chemoselective immobilization of peptides to achieve high density spots with the advantages of fluorescence-based analysis of phosphorylation for nonhazardous detection. For detection of on-chip phosphorylation, we used a fluorescence-labeled antiphosphotyrosine antibody to detect phosphotyrosine and a biotinylated Phostag, which was subsequently bound with a fluorescence-labeled streptavidin for phosphoserine/threonine. More than 290 kinds of Tyr peptides and over 1,100 kinds of Ser/Thr peptides were chemoselectively immobilized onto a glass surface in a high-density format to profile a panel of protein kinases, including c-Src, c-Abl, EGFR, JNK1, ERK2, p38α, and PKA. Many novel, highly reactive and specific peptides were identified as substrates for each protein kinase. Most substrates had the consensus motifs that have been reported previously but some new motifs were also found. The identification of two designed peptides that have higher reactivity than the famous PKA substrate (Kemptide) indicates that analysis of the amino acid biases of substrates is very helpful to the design of new substrates with high reactivity. Thus, the high-density peptide microarray is expected to be a powerful approach for high-throughput discovery of potential substrates for protein kinases.

Signal-to-noise ratio improvement of peptide microarrays by using hyperbranched-polymer materials

The fabrication of peptide microarrays using hyperbranched polymers (HBPs) to improve the signal-to-noise ratio was demonstrated. Due to a high density of reactive groups at the chain ends of the HBPs, as well as to their spherical shape, HBPs can be used as linkers to increase the amount of immobilized peptides through raising the specific surface area of the glass substrate. A zwitterionic HBP was used as a blocking agent to reduce the noise level of the peptide microarrays. The zwitterionic HBP shows comparably excellent blocking ability to a commercially available BSA-based blocking agent. Thus, it was concluded that HBPs have high potential for the fabrication of highly sensitive peptide microarrays.

Development of a fluorescence peptide chip for the detection of caspase activity.

Proteases play a key role in cell functions, and it is very important to monitor their activities for drug screening and diagnosis of diseases. In the present study, a new class of fluorescence probe, into which a fluorophore and a quencher have been introduced, was developed and applied to the on-chip detection of caspase-3 activity. This probe is non fluorescent in the absence of caspase-3. However, when it is treated with active caspase-3, the fluorescence intensity increases dependent on the caspase-3 activity due to the cleavage of the quencher-containing moiety on a glass slide. This caspase-dependent increase in the fluorescence intensity was also detected when the glass slide immobilizing the probe peptide was treated with cell lysate stimulated by staurosporine (STP), which is an apoptosis-inducing agent. On the other hand, such an increase was not detected in the case of control cell lysate without STP-stimulation. The developed system is a rapid and sensitive method and is useful for the direct measurement of protease activity on a glass array.

A new method for evaluation of intracellular protein kinase signals using mass spectrometry

Abstract Recently, comprehensive analysis in genome or proteome have attracted a lot of interest to many researchers in pharmacology, because of its useful information, such as expression profile of DNA, RNA and protein, to understand physiological events. However, it has not been possible to completely understand the cellular function using such information, because genes and proteins express their functions through extremely complicated interaction. On the other hand, total profile of the intracellular signals is expected to provide more detailed information to understand physiological events because various cellular functions are regulated directly by intracellular signals. We describe here an approach for the convenient and sensitive evaluation of intracellular protein kinase signals using mass spectroscopy. The method is based on a class of new peptide reagents and MALDI–TOF mass spectrometry. Using this system, activity changes in protein kinase A with a dosage of various pharmacological drugs into PC–12 cell were evaluated. These activity changes were found to have good correlation with the results of CREB-regulated gene expression, which was delivered into the cell line. We also evaluated the activity of protein kinase C and Src. This method can easily obtain the profile of many protein kinase activities and be useful for high throughput estimation of intracellular signalings, which is important to drug screening or evaluation of gene function.

Establishment of screening system toward discovery of kinase inhibitors using label-free on-chip phosphorylation assays

We describe a label-free method for the kinase inhibition assay toward discovery of kinase inhibitors. The surface plasmon resonance (SPR) imaging analysis using zinc(II) compound was adopted on the on-chip phosphorylation analysis. In this study, following three subjects were focused: (1) to monitor the inhibition of three inhibitors supporting by their specific inhibition mechanisms, (2) to quantify the inhibitory activities, and (3) to prove the reliability of the obtained 50% inhibition concentration (IC(50)) value. First, the inhibitory activities of Amide 5-24, H-89 and Gö6983 on PKA and PKCdelta were determined, and specific inhibitions for two kinases could be observed quantitatively. Second, the inhibition curves of Amide 5-24, Amide 14-22 and H-89 were obtained, and the results supported the two previous reports: (1) the inhibition efficiency of Amide 5-24 was much higher than that of Amide 14-22, and (2) the inhibitory activity of H-89 followed ATP-binding site blocking mechanism. Last, the obtained IC(50) values by the SPR imaging were almost corresponded to those by the solution assay, although on-chip phosphorylation efficiency was low (approximately 12%). In conclusion, validation of the on-chip phosphorylation analysis for kinase inhibitors was achieved. This label-free method might be applied for discovery of kinase inhibitors.