Myung Kyu Lee

A novel mode of action of YC-1 in HIF inhibition: stimulation of FIH-dependent p300 dissociation from HIF-1{alpha}.

Hypoxia-inducible factor (HIF)-1 plays a key role in tumor promotion by inducing ∼60 genes required for tumor adaptation to hypoxia; thus, it is viewed as a target for cancer therapy. For this reason, YC-1, which down-regulates HIF-1α and HIF-2α at the post-translational level, is being developed as a novel anticancer drug. We here found that YC-1 acts in a novel manner to inhibit HIF-1. In the Gal4 reporter system, which is not degraded by YC-1, YC-1 was found to significantly inactivate the COOH-terminal transactivation domain (CAD) of HIF-1α, whereas it failed to inactivate CAD(N803A) mutant. In coimmunoprecipitation assays, YC-1 stimulated factor inhibiting HIF (FIH) binding to CAD even in hypoxia, whereas it failed to increase the cellular levels of hydroxylated Asn803 of CAD. It was also found that YC-1 prevented p300 recruitment by CAD in mammalian two-hybrid and coimmunoprecipitation assays. The involvement of FIH in YC-1-induced CAD inactivation was confirmed in EPO-enhancer and Gal4 reporter systems using FIH small interfering RNA and dimethyloxalylglycine FIH inhibitor. Indeed, FIH inhibition rescued HIF target gene expressions repressed by YC-1. In cancer cell lines other than Hep3B, YC-1 inhibits HIF-1α via the FIH-dependent CAD inactivation as well as via the protein down-regulation. Given these results, we suggest that the functional inactivation of HIF-α contributes to the YC-1-induced deregulation of hypoxia-induced genes. [Mol Cancer Ther 2008;7(12):3729–38]

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor‐type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPρ is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto‐domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto‐domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane–proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain‐specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

Cecropin a ‐ magainin 2 hybrid peptides having potent antimicrobial activity with low hemolytic effect

In order to obtain peptides having improved antimicrobial activity with low hemolytic effect, a hybrid peptide (CA‐MA) composed from cecropin A (1‐8) and magainin 2(1‐12), and its analogues with amino acid substitutions were designed and synthesized. The antimicrobial activities against bacterial cells and hemolytic activities against human red blood cells were analyzed for each peptide. Secondary structures of the peptides in aqueous solution, 50% trifluoroethanol, and sodium dodecylsulfate micelles were estimated using circular dichroism spectroscopy. The increase in hydrophobicity or α‐helicity of the peptides correlated with an increase in hemolytic activity rather than antimicrobial activity. The substitution of Leu for Ser at position 16 in CA‐MA resulted in a remarkable increase in antimicrobial activity without a significant change in hemolytic activity. Furthermore, the increase in antimicrobial activity of the peptides was not always accompanied by the increase in hemolytic activity.

Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1α

The interplay among hypoxia-inducible factor 1-alpha (HIF-1alpha), p53 and human orthologue of murine double minute 2 (Hdm2) has been introduced as a key event in tumor promotion and angiogenesis. Recently, nutlin-3, a small-molecule antagonist of Hdm2, was demonstrated to inhibit the HIF-1-mediated vascular endothelial growth factor production and tumor angiogenesis. Yet, the mechanism by which nutlin-3 inhibits HIF-1 is an open question. We here addressed the mode-of-action of nutlin-3 with respect to the HIF-1alpha-p53-Hdm2 interplay. The effect of nutlin-3 on HIF-1alpha function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1alpha, which occurred p53-dependently but von Hippel-Lindau-independently. On the contrary, nutlin-3 blunted the hypoxic induction of vascular endothelial growth factor by inactivating HIF-1 even in p53-null cells. The C-terminal transactivation domain (CAD) of HIF-1alpha was inactivated by nutlin-3, and furthermore, the factor-inhibiting hypoxia-inducible factor (FIH) hydroxylation of Asn803 was required for the nutlin-3 action. In terms of protein interactions, Hdm2 competed with FIH in CAD binding and inhibited the Asn803 hydroxylation both in vivo and in vitro, which facilitated p300 recruitment. Moreover, nutlin-3 reinforced the FIH binding and Ans803 hydroxylation by inhibiting Hdm2. In conclusion, Hdm2 functionally activates HIF-1 by inhibiting the FIH interaction with CAD, and the Hdm2 inhibition by nutlin-3 results in HIF-1 inactivation and vascular endothelial growth factor suppression. The interplays among HIF-1alpha, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1alpha.

Design of novel analogue peptides with potent fungicidal but low hemolytic activity based on the cecropin a‐melittin hybrid structure

In order to design synthetic peptides with potent antifungal activity but low cytotoxic activity under physiological conditions, several analogues of the previously reported cecropin A (CA)‐melittin (ME) hybrid peptide, CA(1‐8)‐ME(1‐12), were synthesized. These analogues were designed by analysis of the α‐helical wheel diagram of CA(1‐8)‐ME(1‐12). Antifungal activities were measured by growth inhibition of the yeast Trichosporon beigelii and by hemolytic assay with human red blood cells, respectively. Substitution of Thr for Lys at position 18 and 19 of CA(1‐8)‐ME(1‐12) caused a dramatic reduction in hemolytic activity. Two analogue peptides (analogue I and III) showed more potent antifungal and lower hemolytic activity than the original peptide. To study the antifungal mechanism of these peptides, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed with the most powerful antifungal analogue I peptide designed in the present study. As determined by propidium iodide staining, fungal cells treated with analogue I or melittin showed higher fluorescence intensity than those treated with the weak antifungal peptide, cecropin A. By confocal microscopy the analogue I was detected in the intracellular region as well as the in cell membrane. These facts suggested that the antifungal function of this novel peptide analogue acts by pore formation in the cell membrane.

Inhibitor of nuclear factor-kappaB alpha derepresses hypoxia-inducible factor-1 during moderate hypoxia by sequestering factor inhibiting hypoxia-inducible factor from hypoxia-inducible factor 1α

Hypoxia and inflammation often develop concurrently in numerous diseases, and both hypoxia‐inducible factor (HIF)‐1α and nuclear factor‐kappaB (NF‐κB) are key transcription factors of stress response genes. An NF‐κB inhibitor, inhibitor of NF‐κBα (IκBα), was found to interact with factor inhibiting HIF (FIH) and to be hydroxylated by FIH. However, FIH did not functionally regulate IκBα, and the consequence of the FIH–IκBα interaction thus remains uncertain. In the present study, we tested the possibility that IκBα regulates FIH. FIH–IκBα binding was confirmed by yeast two‐hybrid and coimmunoprecipitation analyses. Functionally, IκBα expression further enhanced the transcriptional activity of HIF‐1α under hypoxic conditions. Furthermore, IκBα knockdown repressed HIF‐1α activity. Mechanistically, IκBα derepressed HIF‐1α activity by inhibiting the FIH‐mediated Asn803 hydroxylation of HIF‐1α. It was also found that IκBα activated HIF‐1α by sequestering FIH from HIF‐1α. However, the effect of IκBα on HIF‐1α activity was only observed in atmospheres containing 1% or more of oxygen. After tumor necrosis factor‐α treatment, IκBα downregulation, Asn803 hydroxylation and HIF‐1α inactivation all occurred up to 8 h, but subsided later. On the basis of these results, we propose that IκBα plays a positive regulatory role during HIF‐1‐mediated gene expression. Therefore, IκBα, owing to its interactions with NF‐κB and HIF‐1α, may play a pivotal role in the crosstalk between the molecular events that underlie inflammatory and hypoxic responses.

Monoclonal Antibody-Based Screening Assay for Factor Inhibiting Hypoxia-Inducible Factor Inhibitors:

The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1α (HIF-1α), and the modification abrogates the transcriptional activity of HIF-1α. Because FIH is more active on HIF-1α than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1α, specifically to Asn803 hydroxylated HIF-1α and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1α showed 740 times higher affinity to the Asn803 hydroxylated HIF-1α peptide than the unmodified one. An enzyme-linked immunosorbent assay—based system using SHN-HIF1α displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1α also showed an Asn803 hydroxylation-dependent specificity to HIF-1α in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors. (Journal of Biomolecular Screening 2008:494-503)

The relationships between biophysical activity and the secondary structure of synthetic peptides from the pulmonary surfactant protein SP‐B

Synthetic pulmonary surfactants consisting of a mixture of phospholipids with synthetic peptides based on human and bovine surfactant‐associated protein SP‐B were prepared. These surfactants were analyzed for their biophysical activities by Wilhemly balance experiments and for their secondary structures by circular dichroism (CD) spectroscopy. Four synthetic peptides (SP‐1, SP‐2, SP‐3, and SP‐4) combined with the phospholipid mixture displayed significant surfactant properties. The CD spectra showed that the α‐helical propensities of the peptides in SDS micelles were related to their surfactant activities. These results suggested that the several truncated peptides originated from SP‐B protein, when appropriately recombined with phospholipids, could be used as an effective synthetic surfactant for clinical use.

TMPRSS4 upregulates uPA gene expression through JNK signaling activation to induce cancer cell invasion.

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates tumor cell invasion, migration, and metastasis. However, the mechanisms by which TMPRSS4 contributes to invasion are not fully understood. Here, we demonstrated that TMPRSS4 induced the transcription of the urokinase-type plasminogen activator (uPA) gene through activating the transcription factors Sp1, Sp3, and AP-1 in mainly a JNK-dependent manner and that the induction of uPA was required for TMPRSS4-mediated cancer cell invasion and signaling events. In addition, the uPA receptor was involved in TMPRSS4-induced signaling activation and subsequent uPA expression probably through its association with TMPRSS4 on the cell surface. Immunohistochemical analysis showed that uPA expression was significantly correlated with TMPRSS4 expression in human lung and prostate cancers. These observations suggest that TMPRSS4 is an important regulator of uPA gene expression; the upregulation of uPA by TMPRSS4 contributes to invasion and may represent a novel mechanism for the control of invasion.

The use of multiple antigenic peptide (MAP) in the immunodiagnosis of human immunodeficiency virus infection

In order to develop an ELISA system for the antibody detection of HIV‐1 or HIV‐2 infections, MAPs for HIV‐1 gp41(584‐618) and HIV‐2 gp36(574‐602) corresponding to the immunodominant regions of HIV‐1 gp41 and HIV‐2 gp36 were used as coating antigens in the ELISA. The MAPs were synthesized by the solid phase method using Fmoc‐Lys(Fmoc)‐OH and their molecular weights were confirmed by tricine gel electrophoresis. The MAPs reacted with all HIV positive sera (64 samples), but did not react with HIV negative sera (48 samples) obtained from healthy blood donors. The MAPs showed high sensitivity and specificity in anti‐HIV 1/2 combo panel and anti‐HIV‐1 seroconversion panels. The results indicated that the ELISA system using synthetic MAPs of gp41(584‐618) and gp36(574‐602) as coating antigens can be used as an analytical system for the immunodiagnosis of HIV‐1 or HIV‐2 infections.