Sang-Gi Paik

Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer

Toll-like receptor 2 (TLR2) initiates potent immune responses by recognizing diacylated and triacylated lipopeptides. Its ligand specificity is controlled by whether it heterodimerizes with TLR1 or TLR6. We have determined the crystal structures of TLR2-TLR6-diacylated lipopeptide, TLR2-lipoteichoic acid, and TLR2-PE-DTPA complexes. PE-DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, is a synthetic phospholipid derivative. Two major factors contribute to the ligand specificity of TLR2-TLR1 or TLR2-TLR6 heterodimers. First, the lipid channel of TLR6 is blocked by two phenylalanines. Simultaneous mutation of these phenylalanines made TLR2-TLR6 fully responsive not only to diacylated but also to triacylated lipopeptides. Second, the hydrophobic dimerization interface of TLR2-TLR6 is increased by 80%, which compensates for the lack of amide lipid interaction between the lipopeptide and TLR2-TLR6. The structures of the TLR2-lipoteichoic acid and the TLR2-PE-DTPA complexes demonstrate that a precise interaction pattern of the head group is essential for a robust immune response by TLR2 heterodimers.

Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation

B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI and BAFF-R. We have determined the crystal structure of the extracellular domain of BAFF-R bound to BAFF at a resolution of 3.3 Å. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a β-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the β-turn and is indispensable in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. The extracellular domain of TACI contains two CRDs, both of which contain the DxL motif. Modeling of TACI–BAFF complex suggests that both CDRs simultaneously interact with the BAFF dimer in the virus-like cage.

Expression and regulation of NDRG2 (N-myc downstream regulated gene 2) during the differentiation of dendritic cells

We searched for genes with expressions specific to human monocyte‐derived dendritic cells (DCs) using differential display reverse transcription‐polymerase chain reaction, and found that N‐myc downstream regulated gene 2 (NDRG2), a member of a new family of differentiation‐related genes, was expressed in DCs. While DCs derived from CD34+ progenitor cells also showed strong NDRG2 expression, the corresponding mRNA expression was absent in other cell lines including monocytes, B cells, and NK cells. The inhibition of DC differentiation by dexamethasone or vitamin D3 treatment down‐regulated the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine‐conditioned culture. The level of NDRG2 gene expression in DCs was significantly higher than that of other members of the NDRG gene family. Finally, in contrast to the stable NDRG2 expression in CD40‐stimulated DCs, the induction of DC maturation by lipopolysaccharide (LPS) resulted in the down‐regulation of NDRG2 gene expression. This down‐regulation is likely to be due to a modification and subsequent destabilization of NDRG2 mRNA, because co‐treating with actinomycin D and LPS significantly blocked this LPS effect. Taken together, our results indicate that NDRG2 is expressed during the differentiation of DCs, and that NDRG2 gene expression is differentially regulated by maturation‐inducing stimuli.

Peroxisome Proliferators-Activated Receptor (PPAR) Modulators and Metabolic Disorders

Overweight and obesity lead to an increased risk for metabolic disorders such as impaired glucose regulation/insulin resistance, dyslipidemia, and hypertension. Several molecular drug targets with potential to prevent or treat metabolic disorders have been revealed. Interestingly, the activation of peroxisome proliferator-activated receptor (PPAR), which belongs to the nuclear receptor superfamily, has many beneficial clinical effects. PPAR directly modulates gene expression by binding to a specific ligand. All PPAR subtypes (α, γ, and σ) are involved in glucose metabolism, lipid metabolism, and energy balance. PPAR agonists play an important role in therapeutic aspects of metabolic disorders. However, undesired effects of the existing PPAR agonists have been reported. A great deal of recent research has focused on the discovery of new PPAR modulators with more beneficial effects and more safety without producing undesired side effects. Herein, we briefly review the roles of PPAR in metabolic disorders, the effects of PPAR modulators in metabolic disorders, and the technologies with which to discover new PPAR modulators.

dSETDB1 and SU(VAR)3-9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster.

Germline-stem cells (GSCs) produce gametes and are thus true “immortal stem cells”. In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. Here we show that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3–9. Loss-of-function mutations in dsetdb1 or Su(var)3–9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3–9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype.

Up-regulation of Mac-2 binding protein by hTERT in gastric cancer

Mac‐2 binding protein (Mac‐2BP) is a secreted tumor antigen that is elevated in many cancers and implicated in tumor metastasis, as well as cell adhesion and immune functions. We focused on the human telomerase reverse transcriptase (hTERT) induced Mac‐2BP expression and the relationship between Mac‐2BP expression and the progression of gastric cancer. A cDNA expression array analysis was performed on the telomerase‐negative cell line, SW13, which was engineered to overexpress hTERT when compared with the parental SW13 cell. hTERT‐induced Mac‐2BP expression was confirmed via RT‐PCR and Northern blotting. ELISA and flow cytometric analyses revealed that Mac‐2BP protein was increased by 2‐ to 4‐fold in hTERT‐overexpressing cells compared with the mock control. Mac‐2BP expression was significantly reduced when the overexpressed hTERT was neutralized by the introduction of hTERT‐specific siRNA. These results suggest that Mac‐2BP expression is modulated by hTERT. Mac‐2BP levels in both gastric cancer cells and tumor tissues were determined via Northern blot analysis and immunohistochemistry. Mac‐2BP protein was highly expressed in most gastric cancer cell lines, and gastric tumor tissues were stained more densely than normal tissues. The intracellular and secreted Mac‐2BP levels were also evaluated via ELISA, indicating that Mac‐2BP was expressed and secreted more abundantly in gastric cancer patients than in healthy donors. The elevated serum Mac‐2BP level in gastric tumor patients was also significantly associated with distant metastasis (p = 0.05) and higher tumor stage (p = 0.04). Our findings suggest that Mac‐2BP is induced by hTERT, and that it may prove to be a useful prognostic marker for the detection of malignant progression of metastatic stomach cancers.

Progressive neuronal loss and behavioral impairments of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid precursor protein

The beta-secretase cleaved Abeta-bearing carboxy-terminal fragments (betaCTFs) of amyloid precursor protein (APP) in neural cells have been suggested to be cytotoxic. However, the functional significance of betaCTFs in vivo remains elusive. We created a transgenic mouse line Tg-betaCTF99/B6 expressing the human betaCTF99 in the brain of inbred C57BL/6 strain. Tg-betaCTF99/B6 mouse brain at 12-16 months showed severely down-regulated calbindin, phospho-CREB, and Bcl-xL expression and up-regulated phospho-JNK, Bcl-2, and Bax expression. Neuronal cell density in the Tg-betaCTF99/B6 cerebral cortex at 16-18 months was lower than that of the non-transgenic control, but not at 5 months. At 11-14 months, Tg-betaCTF99/B6 mice displayed cognitive impairments and increased anxiety, which were not observed at 5 months. These results suggest that increased betaCTF99 expression is highly detrimental to the aging brain and that it produces a progressive and age-dependent AD-like pathogenesis.

Activation of Ras Up-regulates Pro-apoptotic BNIP3 in Nitric Oxide-induced Cell Death

Nitric oxide (NO) produced by NO synthases causes nitration and nitrosylation of cellular factors. We have shown previously that endogenously produced or exogenously added NO induces expression of BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3), leading to death of macrophages (Yook, Y.-H., Kang, K.-H., Maeng, O., Kim, T.-R., Lee, J.-O., Kang, K.-i., Kim, Y.-S., Paik, S.-G., and Lee, H. (2004) Biochem. Biophys. Res. Commun. 321, 298–305). We now provide evidence that Ras mediates NO-induced BNIP3 expression via the MEK/ERK/hypoxia-inducible factor (HIF)-1 pathway. (a) ras-Q61L, a constitutively active form of Ras, up-regulated BNIP3 protein expression by enhancing Bnip3 promoter activity, and ras-S17N, a dominant-negative form, and ras-C118S, an S-nitrosylation mutant, blocked NO-induced BNIP3 expression, suggesting that Ras acts downstream of NO and that NO activates Ras by nitrosylation. (b) U0126, a specific MEK inhibitor, completely abolished BNIP3 expression and the stimulation of promoter activity by NO and Ras, whereas 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, SB203580, and wortmannin, specific inhibitors of soluble guanylyl cyclase, p38 MAPK, and phosphatidylinositol 3-kinase, respectively, had no effect. Ras, MEK1/2, and ERK1/2 were sequentially activated by NO treatment of macrophages. (c) Mutation of the HIF-1-binding site (hypoxia-response element) in the Bnip3 promoter abolished BNIP3 induction, and HIF-1α was strongly induced by NO. (d) Transient expression of activated Ras promoted macrophage death, as did NO, and this Ras-mediated cell death was inhibited by silencing BNIP3 expression. These results suggest that NO-induced death of macrophages is mediated, at least in part, by BNIP3 induction.

Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation.

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser132 loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.

Cadmium-induced astroglial death proceeds via glutathione depletion.

Cadmium is a heavy metal that accumulates in the body, and its accumulation in the brain damages both neurons and glial cells. In the current study, we explored the mechanism underlying cadmium toxicity in primary cortical astroglia cultures. Chronic treatment with 10 μM cadmium was sufficient to cause 90% cell death in 18 hr. However, unlike that observed in neurons, cadmium‐induced astroglial toxicity was not attenuated by the antioxidants trolox (100 μM), caffeic acid (1 mM), and vitamin C (1 mM). In contrast, extracellular 100 μM glutathione (GSH; γ‐Glu‐Cys‐Gly) or 100 μM cysteine almost completely blocked cadmium‐induced astroglial death, whereas 300 μM oxidized GSH (GSSG) or 300 μM cystine, which do not have the free thiol group, were ineffective. In addition, cadmium toxicity was noticeably inhibited or enhanced when intracellular GSH was, respectively, increased by using the cell‐permeable glutathione ethyl ester (GSH‐EE) or depleted by using buthionine sulfoximine (BSO), an inhibitor of γ‐glutamylcysteine synthetase. In agreement with these data, intracellular GSH levels were found to be depressed in cadmium‐treated astrocytes. These results suggest that the toxic effect of cadmium on primary astroglial cells involves GSH depletion and, furthermore, that GSH administration can potentially be used to counteract cadmium‐induced astroglial cell death therapeutically.