Hyo Jin Kang

Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity

Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level.

Improvement of the compressive strength of a cuttlefish bone-derived porous hydroxyapatite scaffold via polycaprolactone coating

Cuttlefish bones (CBs) have emerged as attractive biomaterials because of their porous structure and components that can be converted into hydroxyapatite (HAp) via a hydrothermal reaction. However, their brittleness and low strength restrict their application in bone tissue engineering. Therefore, to improve the compressive strength of the scaffold following hydrothermal conversion to a HAp form of CB (CB-HAp), the scaffold was coated using a polycaprolactone (PCL) polymer at various concentrations. In this study, raw CB was successfully converted into HAp via a hydrothermal reaction. We then evaluated their surface properties and composition by scanning electron microscopy and X-ray diffraction analysis. The CB-HAp coated with PCL showed improved compressive performance and retained a microporous structure. The compressive strength was significantly increased upon coating with 5 and 10% PCL, by 2.09- and 3.30-fold, respectively, as compared with uncoated CB-HAp. However, coating with 10% PCL resulted in a reduction in porosity. Furthermore, an in vitro biological evaluation demonstrated that MG-63 cells adhered well, proliferated and were able to be differentiated on the PCL-coated CB-HAp scaffold, which was noncytotoxic. These results suggest that a simple coating method is useful to improve the compressive strength of CB-HAp for bone tissue engineering applications.

Comparison of in vitro and in vivo bioactivity: cuttlefish-bone-derived hydroxyapatite and synthetic hydroxyapatite granules as a bone graft substitute

Bone reconstruction in clinical settings often requires bone substitutes. Hydroxyapatite (HAp) is a widely used bone substitute due to its osteoconductive properties and bone bonding ability. The aim of this study was to evaluate HAp granules derived from cuttlefish bone (CB-HAp) as a substitute biomaterial for bone grafts. In this study, HAp granules were prepared from raw CB by using a hydrothermal reaction. The formation of HAp from CB was confirmed by scanning electron microscopy and x-ray diffraction analysis. The bioactivity of the CB-HAp granules was evaluated both in vitro and in vivo. Our results show that CB-HAp is non-toxic and that CB-HAp granules supported improved cell adhesion, proliferation and differentiation compared to stoichiometric synthetic HAp granules. Furthermore, in vivo bone defect healing experiments show that the formation of bone with CB-HAp is higher than that with pure HAp. These results show that CB-HAp granules have excellent potential for use as a bone graft material.

Homogeneous detection of caspase-3 using intrinsic fluorescence resonance energy transfer (iFRET).

Caspase-3 is an apoptotic cysteine protease and its aberrancy is highly implicated to numerous diseases thereby rendering caspase-3 activity as an important disease marker. Most caspase-3 sensors are caspase-3 substrates of which the fluorescence signals are turned on upon catalytic cleavage by active caspase-3. However, once the signal is generated, the fluorescence does not disappear albeit caspase-3 activity is abolished. Recently, we and other groups have developed the intrinsic Förster resonance energy transfer (iFRET) technique, which utilizes tryptophan residues of the target proteins and target-specific probes, as FRET donors and acceptors, respectively. Due to this principle, iFRET does not require the labeling of target proteins. In this work, we report the development of caspase-3 specific iFRET probes by structure-based design and synthesis, and the successful detection of caspase-3 in cell lysates as well as in its purified form. The limit-of-detection (LOD) of the probes in case of purified caspase-3 was found to be 1.4-1.5 nM. The designed probes did not bind to either procaspase-3 or C163S caspase-3, which are catalytic inactive, confirming that the observed iFRET signal correlates to the catalytic activity of caspase-3. Furthermore, in competition experiments with Ac-DEVD-CHO, a known competitive inhibitor of caspase-3, the iFRET signal was inhibited.

Identification of sennoside A as a novel inhibitor of the slingshot (SSH) family proteins related to cancer metastasis

Graphical abstract Figure. No Caption available. ABSTRACT Phospho‐cofilin (p‐cofilin), which has a phosphate group on Ser‐3, is involved in actin polymerization. Its dephosphorylated form promotes filopodia formation and cell migration by enhancing actin depolymerization. Protein phosphatase slingshot homologs (SSHs), known as dual‐specificity phosphatases, catalyze hydrolytic removal of the Ser‐3 phosphate group from phospho‐cofilin. Aberrant SSH activity results in cancer metastasis, implicating SSHs as potential therapeutic targets for cancer metastasis. In this study, we screened 658 natural products purified from traditional oriental medicinal plants to identify three potent SSH inhibitors with submicromolar or single‐digit micromolar Ki values: gossypol, hypericin, and sennoside A. The three compounds were purified from cottonseed, Saint John’s wort, and rhubarb, respectively. Sennoside A markedly increased cofilin phosphorylation in pancreatic cancer cells, leading to impaired actin dynamics in pancreatic cancer cells with or without EGF stimulation and reduced motility and invasiveness in vitro and in vivo. Collaboratively, these results demonstrate that sennoside A is a novel inhibitor of SSHs and suggest that it may be valuable in the development of pharmaceutical drugs for treating cancer metastasis.

Cyclic peptide ligand with high binding capacity for affinity purification of immunoglobulin G.

The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has necessitated the development of antibody production and purification technologies for both academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the present study we describe the development of a reusable high affinity column for antibody purification utilizing immobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins, packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Ser one showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab). The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than was required for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swine human sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in the present study.

IgG Fc-binding peptide (FcBP)-tat conjugate as a smart antibody carrier into live cells

While use of RNA interference results in total loss of the target proteins, antibody binds to the corresponding epitope region but does not affect the expression level of the target protein. Consequently, it may offer opportunity to selectively evaluate biological function of selected domains from multi-domain proteins where each domain participates in its unique biological function. Although several methods for antibody transduction into live cells have emerged, most of them are suffered from harsh conditions or low delivery efficiency. In this study, we developed antibody transducer by conjugating IgG Fc-binding peptide (FcBP) with Tat, a cell penetrating peptide (CPP). FcBPCPP fusion peptide and FcBP-eGFP-Tat fusion protein, thus prepared, self-assembled to IgGs in situ upon their simple mixing in cell growth media, and translocated into the cytosol of live cells. Since FcBP-CPP reversibly binds to Fc-region of antibodies it neither impairs the structural and functional integrity of antibodies nor requires additional effort to modify antibodies. This represents the first synthetic peptide reagent, enabling antibody delivery into live cells in the absence of any additional detergents or other physical treatments such as electrophoresis or microinjection.

Label-free detection of protein tyrosine phosphatase 1B (PTP1B) using a rationally designed Förster resonance energy transfer (FRET) probe

A highly selective detection method of native protein tyrosine phosphatase 1B (PTP1B) is described using a target specific probe equipped with 1‐naphthylamine (λex=330 nm, λem=445 nm). Irradiation of a mixture of PTP1B and Probe 1 with ultraviolet light of 280 nm (corresponding to PTP1B excitation maximum) resulted in significant fluorescence increase at 445 nm, following FRET characteristics. This phenomenon does not occur with other closely related phosphatases or cellular abundant alkaline phosphatase (APP). Probe 1, the most potent and selective probe, was found to competitively inhibit PTP1B (Ki≈42 nm), whereas APP inhibition was found to be in the low micromolar range. Furthermore, Probe 1 discriminates between PTP1B and several other phosphatases. Here, we report real‐time label‐free FRET detection of pure PTP1B as well as induced human PTP1B in Escherichia coli cell lysate. In contrast to 6,8‐difluoro‐4‐methylumbelliferyl phosphate (DiFMUP), a representative fluorescence turn‐on PTP substrate, our FRET probe successfully differentiated human cervical carcinoma cell lysate, SiHa, which has a high expression level of PTP1B, from PTP1B‐knockdown SiHa cell lysate (that is, siRNA was used for PTP1B knockdown).

Ginkgolic acid as a dual-targeting inhibitor for protein tyrosine phosphatases relevant to insulin resistance

Several protein tyrosine phosphatases (PTPs) that disrupt the insulin-signaling pathway were investigated by siRNAs to identify potential antidiabetic targets. Individual knockdown of PTPN9 and DUSP9 in 3T3-L1 preadipocytes increased AMPK phosphorylation, respectively, and furthermore, concurrent knockdown of both PTPN9 and DUSP9 synergistically increased AMPK phosphorylation. Next, 658 natural products were screened to identify dual inhibitors of both PTPN9 and DUSP9. Based on the selectivity and inhibition potency of the compounds, ginkgolic acid (GA) was selected for further study as a potential antidiabetic drug candidate. GA inhibited the enzymatic activity of PTPN9 (Ki = 53 µM) and DUSP9 (Ki = 2.5 µM) in vitro and resulted in a significant increase of glucose-uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes. In addition, GA increased phosphorylation of AMPK in 3T3L1 adipocytes. In this study, GA as a dual targeting inhibitor of PTPN9 and DUSP9 increased glucose uptake in 3T3L1 and C2C12 cells by activating the AMPK signaling pathway. These results strongly suggest GA could be used as a therapeutic candidate for type 2 diabetes.