Sun Joo Park

Phosphatidylinositol 4-Phosphate 5-Kinase Type I Is Regulated through Phosphorylation Response by Extracellular Stimuli

Phosphatidylinositol 4-phosphate 5-kinase (PIPK) catalyzes a final step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid signaling molecule. Strict regulation of PIPK activity is thought to be essential in intact cells. Here we show that type I enzymes of PIPK (PIPKI) are phosphorylated by cyclic AMP-dependent protein kinase (PKA), and phosphorylation of PIPKI suppresses its activity. Serine 214 was found to be a major phosphorylation site of PIPK type Iα (PIPKIα) that is catalyzed by PKA. In contrast, lysophosphatidic acid-induced protein kinase C activation increased PIPKIα activity. Activation of PIPKIα was induced by dephosphorylation, which was catalyzed by an okadaic acid-sensitive phosphatase, protein phosphatase 1 (PP1). In vitro dephosphorylation of PIPKIα with PP1 increased PIPK activity, indicating that PP1 plays a role in lysophosphatidic acid-induced dephosphorylation of PIPKIα. These results strongly suggest that activity of PIPKIα in NIH 3T3 cells is regulated by the reversible balance between PKA-dependent phosphorylation and PP1-dependent dephosphorylation.

Interaction of HSP90 to N-WASP leads to activation and protection from proteasome-dependent degradation.

Neural Wiskott–Aldrich syndrome protein (N‐WASP) regulates reorganization of the actin cytoskeleton through activation of the Arp2/3 complex. Here, we show that heat shock protein 90 (HSP90) regulates N‐WASP‐induced actin polymerization in cooperation with phosphorylation of N‐WASP. HSP90 binds directly to N‐WASP, but binding alone does not affect the rate of N‐WASP/Arp2/3 complex‐induced in vitro actin polymerization. An Src family tyrosine kinase, v‐Src, phosphorylates and activates N‐WASP. HSP90 increases the phosphorylation of N‐WASP by v‐Src, leading to enhanced N‐WASP‐dependent actin polymerization. In addition, HSP90 protects phosphorylated and activated N‐WASP from proteasome‐dependent degradation, resulting in amplification of N‐WASP‐dependent actin polymerization. Association between HSP90 and N‐WASP is increased in proportion to activation of N‐WASP by phosphorylation. HSP90 is colocalized and associated with active N‐WASP at podosomes in 3Y1/v‐Src cells and at growing neurites in PC12 cells, whose actin structures are clearly inhibited by blocking the binding of HSP90 to N‐WASP. These findings suggest that HSP90 induces efficient activation of N‐WASP downstream of phosphorylation signal by Src family kinases and is critical for N‐WASP‐dependent podosome formation and neurite extension.

Dieckol from Ecklonia cava suppresses the migration and invasion of HT1080 cells by inhibiting the focal adhesion kinase pathway downstream of Rac1-ROS signaling

We have previously isolated dieckol, a nutrient polyphenol compound, from the brown alga, Ecklonia cava (Lee et al., 2010a). Dieckol shows both antitumor and antioxidant activity and thus is of special interest for the development of chemopreventive and chemotherapeutic agents against cancer. However, the mechanism by which dieckol exerts its antitumor activity is poorly understood. Here, we show that dieckol, derived from E. cava, inhibits migration and invasion of HT1080 cells by scavenging intracellular reactive oxygen species (ROS). H2O2 or integrin signal-mediated ROS generation increases migration and invasion of HT1080 cells, which correlates with Rac1 activation and increased expression and phosphorylation of focal adhesion kinase (FAK). Rac1 activation is required for ROS generation. Depletion of FAK by siRNA suppresses Rac1-ROS-induced cell migration and invasion. Dieckol treatment attenuated intracellular ROS levels and activation of Rac1 as well as expression and phosphorylation of FAK. Dieckol treatment also decreases complex formation of FAK-Src-p130Cas and expression of MMP2, 9, and 13. These results suggest that the Rac1-ROS-linked cascade enhances migration and invasion of HT1080 cells by inducing expression of MMPs through activation of the FAK signaling pathway, whereas dieckol downregulates FAK signaling through scavenging intracellular ROS. This finding provides new insights into the mechanisms by which dieckol is able to suppress human cancer progresssion and metastasis. Therefore, we suggest that dieckol is a potential therapeutic agent for cancer treatment.

Hepatoprotective effects of dieckol-rich phlorotannins from Ecklonia cava, a brown seaweed, against ethanol induced liver damage in BALB/c mice

Alcoholic liver disease, which is one of the most serious liver disorders, has been known to cause by ethanol intake. In the present study, in vivo hepatoprotective effects of dieckol-rich phlorotannins (DRP) from Ecklonia cava, a brown seaweed, on ethanol induced hepatic damage in BALB/c mice liver were investigated. After administration of 5 and 25mg/kg mouse of DRP and 4 g/kg mice ethanol, the body weights and survival rates were increased as compared to the control, which is ethanol-treated group without DRP. The glutamic oxaloacetic transaminase and glutamic pyruvic transaminase levels in the serum were lower than those of the control. DRP exhibited a reduction of the total cholesterol. The lower levels of SOD enzyme and a reduction of the formation of malondialdehyde were occurred in mice fed with 5 and 25mg/kg mouse of DRP. Finally the effect on improvement of fatty liver induced by ethanol was observed by taking out the liver immediately after dissecting the mouse. However, no significant difference was observed on hepatic histopathological changes. In conclusion, this study indicated that DRP could protect liver injury induced by ethanol in vivo. It suggested that DRP possesses the beneficial effect to human against ethanol-induced liver injury.

Antioxidant Dieckol Downregulates the Rac1/ROS Signaling Pathway and Inhibits Wiskott-Aldrich Syndrome Protein (WASP)-Family Verprolin-Homologous Protein 2 (WAVE2)-Mediated Invasive Migration of B16 Mouse Melanoma Cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H2O2 treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H2O2-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.

Production of α-amylase for the biosynthesis of gold nanoparticles using Streptomyces sp. MBRC-82

Marine actinobacterial synthesis of gold nanoparticles has good potential to develop simple, cost-effective and eco-friendly methods for production of important biomaterials. In this context, gold nanoparticles have attracted considerable attention in recent years, owing to their various applications. In this paper, we report on the production of α-amylase for the extracellular synthesis of gold nanoparticles using Streptomyces sp. MBRC-82. Medium composition and culture conditions for α-amylase production were statistically optimized. Plackett-Burman design was employed to find out the optimal medium constituents and culture conditions to enhance α-amylase production. Box-Behnken design revealed that three independent variables namely soluble starch (5.8484 g), peptone (3.5191 g), and NaCl (0.3829) significantly influenced α-amylase production. The gold nanoparticles were characterized by ultraviolet-visible (UV-vis) spectrometer, X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDXA), and transmission electron microscopy (TEM). The particles synthesized using the optimized enzyme activity ranged from 20 to 80 nm with an average particle size of 40 nm and therefore can be extended to various medicinal applications.

Nitric oxide-mediated vasorelaxation effects of anti-angiotensin I-converting enzyme (ACE) peptide from Styela clava flesh tissue and its anti-hypertensive effect in spontaneously hypertensive rats.

In our previous study, an anti-angiotensin I converting enzyme (ACE) peptide (Ala-His-Ile-Ile-Ile, MW: 565.3Da) was isolated from Styela clava flesh tissue. In this study the fractions obtained during the isolation process and the finally purified peptide were examined to see if they had vasorelaxation effects in isolated rat aortas, and then the peptide was investigated for anti-hypertensive effect in spontaneously hypertensive rats (SHRs). The induction of vasorelaxation in the rat aortas was observed with the isolated fractions and the peptide from the enzymatic hydrolysate of S. clava flesh tissue and could be markedly blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME). In human endothelial cells, NO synthesis was found to be increased and eNOS phosphorylation was upregulated when the cells were cultured with the purified peptide. Furthermore, systolic blood pressure was reduced by administration of the potent vasorelaxation peptide in SHRs.

HSP90 cross‐links branched actin filaments induced by N‐WASP and the Arp2/3 complex

N‐WASP induces filopodial actin cytoskeleton through activation of the Arp2/3 complex. Here, we show that heat shock protein 90 (HSP90) regulates the structure of actin filaments induced by N‐WASP and the Arp2/3 complex. HSP90 binds to N‐WASP and to F‐actin and bundles actin filaments. Bundling activity of HSP90 does not affect actin filament nucleation induced by N‐WASP and the Arp2/3 complex. HSP90 is co‐localized with N‐WASP at branching points of actin filaments produced by the Arp2/3 complex and thereby bundles branched filaments; this bundled actin structure is inhibited by blocking direct binding between HSP90 and N‐WASP. Furthermore, HSP90 converts branched actin filaments on N‐WASP‐coated beads to filopodia‐like star‐shaped bundles. These findings indicate that HSP90 promotes the formation of N‐WASP/Arp2/3 complex‐induced unbranched filopodial actin structures.

Gallic Acid-g-Chitosan Modulates Inflammatory Responses in LPS-Stimulated RAW264.7 Cells Via NF-κB, AP-1, and MAPK Pathways.

Chitosan is a naturally occurring polysaccharide, which has exhibited antioxidant, antimicrobial, and anti-cancer activities among others. Modification of chitosan by grafting phenolic compounds is a good strategy for improvement of bioactivities of chitosan. We investigated the anti-inflammatory action of gallic acid-grafted-chitosan (GAC) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. GAC inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) by inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 macrophages. GAC also suppressed the production and mRNA expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). GAC inactivated nuclear factor-κB (NF-κB) via inhibiting the phosphorylation and degradation of the NF-κB inhibitor, IκB. In addition, GAC suppresses the activation of activator protein-1 (AP-1) through the phosphorylation of mitogen-activated protein kinase (MAPK) such as extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). These results suggest that GAC has the potential anti-inflammatory action by downregulating transcriptional factors (NF-κB and AP-1) through MAPK signaling pathways.

β-secretase inhibitory activity of phenolic acid conjugated chitooligosaccharides

Eight kinds of phenolic acid conjugated chitooligosaccharides (COSs) were synthesized using hydroxyl benzoic acid and hydroxyl cinnamic acid. These phenolic acid conjugated-COSs with different substitution groups, including p-hydroxyl, 3,4-dihydroxyl, 3-methoxyl-4-hydroxyl and 3,5-dimethoxyl-4-hydroxy groups, were evaluated for their inhibitory activities against β-site amyloid precursor protein (APP)-cleaving enzyme (BACE) and inhibited BACE with a ratio of 50.8%, 74.8%, 62.1%, 64.8% and 42.6%, respectively at the concentration of 1,000 μg/mL. BACE is a critical component to reduce the levels of Aβ amyloid peptide in Alzheimer’s disease (AD) which is based on the amyloid cascade theory in the brain, as this protease initiates the first step in Aβ production. Among them, Caffeic acid conjugated-COS (CFA-COS) was further analysed to determine mode of inhibition of BACE and it showed non-competitive inhibition. Hence in this study, we suggest that CFA-COS derivatives have potential to be used as novel BACE inhibitors to reduce the risk of AD.