Taek Jin Kang

Sonic hedgehog intradermal gene therapy using a biodegradable poly(β-amino esters) nanoparticle to enhance wound healing.

Biodegradable cationic poly(β-amino esters) (PBAE) nanoparticles are promising tools for delivering genes into various types of cells and tissues. Specific end-modification of the PBAE terminal parts significantly improves the efficiency of gene delivery in vitro and in vivo, and reduces cytotoxicity. Here, we demonstrated that amine end-modified PBAE nanoparticles can be used for intradermal delivery of therapeutic genes for wound healing in an animal skin wound model. Sonic hedgehog (SHH), a prototypical morphogen with angiogenic potential, was applied as a therapeutic gene to regenerate skin tissue. Amine end-modified PBAEs showed higher gene transfection efficiency in vitro than the commercial reagent, Lipofectamine 2000. Intradermal delivery of the SHH gene using amine end-modified PBAEs was tested in a readout mouse model of SHH signaling. We evaluated its therapeutic efficacy in mice with full-thickness skin wounds. SHH gene therapy significantly increased the expression of the angiogenic growth factor, vascular endothelial growth factor, and the stromal cell-derived factor-1α chemokine within the wounded regions early after injection. Ultimately, wound closure was accelerated in mice receiving the PBAE/SHH gene therapy compared to mice receiving intradermal delivery of a control gene (β-galactosidase plasmid) by PBAE nanoparticles. Quantitative real-time polymerase chain reaction and histological analysis revealed that there were significant improvements in epidermis regeneration and blood vessel formation in the mice treated with PBAE/SHH nanoparticles. In conclusion, SHH intradermal gene therapy using biodegradable PBAE nanoparticles is a potential treatment to promote wound healing.

Expression of Histone H3 Tails with Combinatorial Lysine Modifications under the Reprogrammed Genetic Code for the Investigation on Epigenetic Markers

We report the ribosomal synthesis of N-terminal peptides of histone H3, so-called H3 tail (H3t), with combinatorial methyl and acetyl modifications of selected lysine residues, and the application of such peptides to studying the influence of lysine modification on H3t binding to chromodomain of heterochromatin protein 1 (chromoHP1). Genetic code reprogramming was employed to reassign four codons to acetylated, mono-, di-, and trimethylated lysines, and 38-mer H3t peptides containing modified lysines at designated sites were expressed from the corresponding mRNA sequences. Using a series of H3t constructs, we show complex crosstalk among methylated lysine 9 and 27, and acetylated lysine 14 for binding to chromoHP1. This proof-of-concept study offers a unique means for the synthesis of not only an H3t library containing modified lysines but also other classes of peptides bearing posttranslational methylation and acetylation.

Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness.

Bacterial glutamate decarboxylase (GAD) transforms glutamate into γ-aminobutyric acid (GABA) with the consumption of a proton. The enzyme is active under acidic environments only and sharply loses its activity as pH approaches neutrality with concomitant structural deformation. In an attempt to understand better the role of this cooperative loss of activity upon pH shifts, we prepared and studied a series of GAD site-specific mutants. In this report, we show that the cooperativeness was kept intact by at least two residues, Glu89 and His465, of which Glu89 is newly identified to be involved in the cooperativity system of GAD. Double mutation on these residues not only broke the cooperativity in the activity change but also yielded a mutant GAD that retained the activity at neutral pH. The resulting mutant GAD that was active at neutral pH inhibited the cell growth in a glycerol medium by converting intracellular Glu into GABA in an uncontrolled manner, which explains in part why the cooperativeness of GAD has to be kept by several layers of safety keepers. This unexpected result might be utilized to convert a low-valued by-product of biodiesel production, glycerol, into value-added product, GABA.

Ribosomal synthesis of nonstandard peptides.

It is well known that standard peptides, which comprise proteinogenic amino acids, can act as specific chemical probes to target proteins with high affinity. Despite this fact, a number of peptide drug leads have been abandoned because of their poor cell permeability and protease instability. On the other hand, nonstandard peptides isolated as natural products often exhibit remarkable pharmaco-behavior and stability in vivo. Although it is likely that numerous nonstandard therapeutic peptides capable of recognizing various targets could have been synthesized, enzymes for nonribosomal peptide syntheses are complex; therefore, it is difficult to engineer such modular enzymes to build nonstandard peptide libraries. Here we describe an emerging technology for the synthesis of nonstandard peptides that employs an integrated system of reconstituted cell-free translation and flexizymes. We summarize the historical background of this technology and discuss its current and future applications to the synthesis of nonstandard peptides and drug discovery.

AIMP3/p18 controls translational initiation by mediating the delivery of charged initiator tRNA to initiation complex.

Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are nonenzymatic scaffolding proteins that comprise multisynthetase complex (MSC) with nine aminoacyl-tRNA synthetases in higher eukaryotes. Among the three AIMPs, AIMP3/p18 is strongly anchored to methionyl-tRNA synthetase (MRS) in the MSC. MRS attaches methionine (Met) to initiator tRNA (tRNA(i)(Met)) and plays an important role in translation initiation. It is known that AIMP3 is dispatched to nucleus or nuclear membrane to induce DNA damage response or senescence; however, the role of AIMP3 in translation as a component of MSC and the meaning of its interaction with MRS are still unclear. Herein, we observed that AIMP3 specifically interacted with Met-tRNA(i)(Met)in vitro, while it showed little or reduced interaction with unacylated or lysine-charged tRNA(i)(Met). In addition, AIMP3 discriminates Met-tRNA(i)(Met) from Met-charged elongator tRNA based on filter-binding assay. Pull-down assay revealed that AIMP3 and MRS had noncompetitive interaction with eukaryotic initiation factor 2 (eIF2) γ subunit (eIF2γ), which is in charge of binding with Met-tRNA(i)(Met) for the delivery of Met-tRNA(i)(Met) to ribosome. AIMP3 recruited active eIF2γ to the MRS-AIMP3 complex, and the level of Met-tRNA(i)(Met) bound to eIF2 complex was reduced by AIMP3 knockdown resulting in reduced protein synthesis. All these results suggested the novel function of AIMP3 as a critical mediator of Met-tRNA(i)(Met) transfer from MRS to eIF2 complex for the accurate and efficient translation initiation.

Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli.

Escherichia coli glutamate decarboxylase (GAD) converts glutamate into γ-aminobutyric acid (GABA) through decarboxylation using proton as a co-substrate. Since GAD is active only at acidic conditions even though pH increases as the reaction proceeds, the conventional practice of using this enzyme involved the use of relatively high concentration of buffers, which might complicate the downstream purification steps. Here we show by simulation and experiments that the free acid substrate, glutamic acid, rather than its monosodium salt can act as a substrate and buffer at the same time. This yielded the buffer- and salt-free synthesis of GABA conveniently in a batch mode. Furthermore, we engineered GAD to hyper active ones by extending or reducing the length of the enzyme by just one residue at its C-terminus. Through the buffer-free reaction with engineered GAD, we could synthesize 1M GABA in 3h, which can be translated into a space-time yield of 34.3g/L/h.

A cell-free protein synthesis system as an investigational tool for the translation stop processes

Using Escherichia coli cell‐free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S‐(2‐nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS–PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell‐free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.

An efficient cell-free protein synthesis system using periplasmic phosphatase-removed S30 extract.

An efficient cell-free translation system was developed by removal of phosphatase localized in the periplasmic space, which hampers the translation reaction by hydrolyzing ATP. S30 extract was prepared from the spheroplast of Escherichia coli, and as much as 40% of ATP-hydrolysis activity of phosphatases could be easily removed by the spheroplast formation. The reaction period of translation using phosphatase-removed S30 extract could be prolonged and protein synthesis was enhanced by more than 30%.

The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli

DNA-binding motifs that are recognized by transcription factors (TFs) have been well studied; however, challenges remain in determining the in vivo architecture of TF-DNA complexes on a genome-scale. Here, we determined the in vivo architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using high-throughput sequencing of exonuclease-treated chromatin-immunoprecipitated DNA (ChIP-exo). The ChIP-exo has a unique peak-pair pattern indicating 5′ and 3′ ends of ArgR-binding region. We identified 62 ArgR-binding loci, which were classified into three groups, comprising single, double and triple peak-pairs. Each peak-pair has a unique 93 base pair (bp)-long (±2 bp) ArgR-binding sequence containing two ARG boxes (39 bp) and residual sequences. Moreover, the three ArgR-binding modes defined by the position of the two ARG boxes indicate that DNA bends centered between the pair of ARG boxes facilitate the non-specific contacts between ArgR subunits and the residual sequences. Additionally, our approach may also reveal other fundamental structural features of TF-DNA interactions that have implications for studying genome-scale transcriptional regulatory networks.

Genetically Engineered Myoblast Sheet for Therapeutic Angiogenesis

Peripheral arterial disease is a common manifestation of systemic atherosclerosis, which results in more serious consequences of ischemic events in peripheral tissues such as the lower extremities. Cell therapy has been tested as a treatment for peripheral ischemia that functions by inducing angiogenesis in the ischemic region. However, the poor survival and engraftment of transplanted cells limit the efficacy of cell therapy. In order to overcome such challenges, we applied genetically engineered cell sheets using a cell-interactive and thermosensitive hydrogel and nonviral polymer nanoparticles. C2C12 myoblast sheets were formed on Tetronic-tyramine (Tet-TA)-RGD hydrogel prepared through a highly efficient and noncytotoxic enzymatic reaction. The myoblast sheets were then transfected with vascular endothelial growth factor (VEGF) plasmids using poly(β-amino ester) nanoparticles to increase the angiogenic potential of the sheets. The transfection increased the VEGF expression and secretion from the C2C12 sheets. The enhanced angiogenic effect of the VEGF-transfected C2C12 sheets was confirmed using an in vitro capillary formation assay. More importantly, the transplantation of the VEGF-transfected C2C12 sheets promoted the formation of capillaries and arterioles in ischemic muscles, attenuated the muscle necrosis and fibrosis progressed by ischemia, and eventually prevented ischemic limb loss. In conclusion, the combination of cell sheet engineering and genetic modification can provide more effective treatment for therapeutic angiogenesis.