Chan Woo Kim

Protein kinase C (PKC) isozyme-specific substrates and their design.

Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, appears to be involved in the signal transduction response to many hormones and growth factors; there are 11 different PKC isozymes. Because PKC isozymes directly and/or indirectly participate in signal transduction pathways of normal and transformed cells through phosphorylation of target proteins, it is critical to understand the diversity of the intracellular signaling pathways regulated by each PKC isozyme. Thus, PKC isozyme-specific substrates are useful to understand the characterization of the intracellular signaling pathways for each PKC isozyme. Consensus sequences and sequence information obtained from PKC target proteins are very important to design PKC isozyme-specific peptide substrates. Moreover, computational prediction programs of phosphorylation sites using a library of peptide substrates aid in the fast design of PKC isozyme-specific peptide substrates. Although a large number of target proteins and synthetic peptides for PKCs are known, only two peptide substrates (peptide 422-426 of murine elongation factor-1α and Alphatomega peptide) have been reported as PKC isozyme-specific peptide substrates. This discussion will review the literature concerning these native and synthetic PKC isozyme-specific peptide substrates and their design.

A protein kinase assay based on FRET between quantum dots and fluorescently-labeled peptides

A novel protein kinase assay was developed, based on FRET between QDs and fluorescently-labeled substrate peptides. The negatively charged QDs recognize the change in net charge of the peptide upon phosphorylation. Despite its simple mechanism, this assay is sensitive and robust enough to be applied to the evaluation of protein kinase inhibitors.

Short peptide motifs for long-lasting anchoring to the cell surface

A rational design strategy has been developed for the construction of stable peptide-based anchors for the efficient modification of cell surfaces. Six types of peptide composed of five residues with divalent hydrophobic groups have been designed using this new strategy. Among them, a peptide with a sequence of NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH2 (NBD: fluorophore, Lys(X): N-ε-palmitoyl-l-lysine) was found to show the highest modification efficacy and longevity in culture medium. The good performance of this peptide was attributed to (1) its high aqueous solubility, which allowed it to partition from the medium to the cell surface, and (2) the high binding affinity of the saturated palmitoyl groups to the cell membrane. We found that the distribution of the peptide was affected by recycling endosome, which enabled the representation of the peptide following its endocytotic disappearance from the cell membrane. Biotin was also presented on the cell surface using this peptide-based anchor to examine its recognition by streptavidin. The efficacy of the recognition process increased as the length of the oligoethylene glycol spacer increased, indicating that it was necessary for the biotin tag to move away from the membrane glycoproteins on the cell surface to facilitate its efficient recognition by streptavidin.

Stabilization of cancer-specific gene carrier via hydrophobic interaction for a clear-cut response to cancer signaling.

Here, we developed a new gene carrier, comprising a linear polyethylenimine (LPEI) grafted with a hydrophobically modified cationic peptide containing a long alkyl chain, for use in cancer-specific gene delivery. The cationic peptide is a substrate of protein kinase Cα (PKCα), which is known to be activated specifically in cancer cells. The hydrophobically modified LPEI-peptide conjugate (LPEI-C10-peptide) could form a polyplex with DNA through electrostatic and hydrophobic interactions between the anionic DNA strands and the cationic peptide substrate. The hydrophobic modification of the peptide did not affect the reactivity of the peptide toward PKCα, while the polyplex showed improved intracellular uptake. Because of the efficient endosomal escape and enhanced stability, the polyplex significantly improved the transgene regulation responding to intracellular PKCα activity.

Histidinylated poly-L-lysine-based vectors for cancer-specific gene expression via enhancing the endosomal escape.

In this work, we synthesized a series of poly-L-lysine (PLL)-based polymers for gene delivery, by modifying the PLL with both cationic peptide and histidine. The peptide moieties serve as cationic centers for polyplex formation, and also as substrates for protein kinase Cα (PKCα), which is specifically activated in many types of cancer cells, to achieve cancer-specific gene expression. The histidine groups serve as buffering moieties to increase the ability of the plasmid DNA (pDNA)-polymer complex (polyplex) to escape the endosome and thus to promote expression of the pDNA in the transfected cells. The facile synthesis of the polymers proceeded by modifying the PLL with side-group-protected peptide and protected histidine, followed by deprotection of the functional groups. The synthesized polymers showed significant buffering capacity over the neutral to acidic pH range and showed less cytotoxicity in vitro compared with histidine-unmodified polymers. The polyplexes successfully showed PKCα-responsive gene expression immediately after their introduction into cancer cells and the gene expression continued for at least 24 h. These PLL-based carriers thus show promise for cancer-targeted gene therapy.

Effect of peptide content on the regulation of transgene expression by protein kinase Cα-responsive linear polyethylenimine-peptide conjugates

We examined a series of linear polyethylenimine (LPEI)-based nanocarriers that activate transgene expression in response to cancer-specific protein kinase Cα (PKCα). Eight types of LPEI-peptide conjugate differing in peptide content and number were synthesized using click chemistry. The conjugates could form polyplexes with pDNA through electrostatic interaction, but the degree of pDNA condensation, sizes, and surface charges of the resulting polyplexes depended on the pendant-peptide content and number. None of the polyplexes showed significant cytotoxicity toward human hepatoma cells (HepG2). Furthermore, pendant peptide content and number markedly affected transgene activation in response to PKCα. To achieve an all-or-none response to PKCα, we determined the optimum peptide content and number in LPEI-peptide conjugates as ≈6 mol% and ≈40 peptides/conjugate.

Cancer-specific gene carriers responding to cancer microenvironment: Acidosis and hyper-activated protein kinases

Protein kinase (PK)-responsive gene carriers modified with polyethylene glycol (PEG) chains using an acid-labile linker were developed. These carriers were obtained by modifying the PEG chains and substrate peptides for the PKs (PKA or PKCα) on the branched polyethyleneimine main chain. Polyplexes formed from these carriers and plasmid DNA (pDNA) were stably dispersed under neutral pH medium. The polyplexes were also taken up by cells on the release of the PEG chains under the slightly acidic extracellular pH associated with cancer cells. The polyplexes taken up by cells resulted in gene expression when the substrate peptides were phosphorylated by the intracellular PKs to release pDNA from the polyplexes. These novel gene carriers are expected to be promising for cancer-specific gene therapy via intravenous administration.

Tumor accumulation of protein kinase-responsive gene carrier/DNA polyplex stabilized by alkanethiol for intravenous injection

We synthesized polymeric gene carriers consisting of poly-L-lysine (PLL) main chain modified both with substrate peptide for protein kinase Cα (PKCα) and alkanethiol (pentadecanethiol). Due to the grafted substrate peptide, the polyplex prepared from these carriers is expected to show gene expression triggered by the phosphorylation of the peptide by intracellular PKCα. The modified alkanethiol on the main chain stabilized the polyplex both via disulfide crosslinking and hydrophobic interaction. The polyplex found to show gene expression in vitro when the alkanethiol content in the main chain was enough low (4-mol%-modification of PLL’s ε-amine group) to minimize cytotoxic effect. Even though the content of alkanethiol is low, the polyplex had significant stability in a model serum solution and showed longer blood circulation in vivo. The polyplex clearly accumulated in tumor after intravenous injection.

Branched polyethylenimine-based PKCα-responsive gene carriers

We examined in vitro performance of the branched polyethylenimine (bPEI)-based gene carriers which respond to cancer-specific activation of protein kinase Cα (PKCα) to express plasmid DNA. The carriers were synthesized straightforward by using amide bond formation between a peptide terminal carboxyl and a primary amine group of bPEI. To examine the effect of the peptide contents in the carrier, we prepared several carriers with various peptide contents. The obtained polymers form polyplexes with tighter condensation of plasmid DNA than our previous gene carriers. After internalization of the polyplexes via endocytosis, the polyplexes effectively escaped from the endosome into cytosol. Then, the polyplexes showed a clear-cut response to PKCα to release plasmid DNA for gene expression. We determined the optimum contents of the peptides in carriers as 5 mol% to achieve the clear-cut response to PKCα.

Utilization of a PNA-peptide conjugate to induce a cancer protease-responsive RNAi effect

Small interfering RNA (siRNA) is regarded as a promising tool for cancer therapy because of the wide applicability to various cancer-related genes. However, non-specific delivery of siRNA is one of the major causes of adverse effects. To access the issue, here we designed a new siRNA system which turns on RNAi responding to a cancer cell-specific protease, cathepsin B. The system uses a peptide nucleic acid (PNA)-peptide conjugate to provide this protease-responsive activation. The PNA-peptides were found to form hybrids with double-stranded RNAs with complementary protruding regions, which then affected the susceptibility of dsRNA to Dicer. The dsRNA/PNA-peptide hybrids were activated in cancer cells with a high cathepsin B activity to show RNAi.