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Yang-Gyun Kim

Publications and source records attributed to Yang-Gyun Kim.

At least 19 recordsLinked to original sources
PubMed2006

A peptide with alternating lysines can act as a highly specific Z-DNA binding domain.

Many nucleic acid binding proteins use short peptide sequences to provide specificity in recognizing their targets, which may be either a specific sequence or a conformation. Peptides containing alternating lysine have been shown to bind to poly(dG-d5meC) in the Z conformation, and stabilize the higher energy form [H. Takeuchi, N. Hanamura, H. Hayasaka and I. Harada (1991) FEBS Lett., 279, 253-255 and H. Takeuchi, N. Hanamura and I. Harada (1994) J. Mol. Biol., 236, 610-617.]. Here we report the construction of a Z-DNA specific binding protein, with the peptide KGKGKGK as a functional domain and a leucine zipper as a dimerization domain. The resultant protein, KGZIP, induces the Z conformation in poly(dG-d5meC) and binds to Z-DNA stabilized by bromination with high affinity and specificity. The binding of KGZIP is sufficient to convert poly(dG-d5meC) from the B to the Z form, as shown by circular dichroism. The sequence KGKGKGK is found in many proteins, although no functional role has been established. KGZIP also has potential for engineering other Z-DNA specific proteins for future studies of Z-DNA in vitro and in vivo.

Amino Acid Sequence↗
PubMed2006

Intracellular localization of human ZBP1: Differential regulation by the Z-DNA binding domain, Zalpha, in splice variants.

We investigated the subcellular distribution of human ZBP1, which harbors the N-terminal Z-DNA binding domains, Zalpha and Zbeta. ZBP1 was distributed primarily in the cytoplasm and occasionally as nuclear foci in interferon (IFN)-treated primary hepatocellular carcinoma cells, and in several other transfected cell types. In leptomycin B (LMB)-treated cells, endogenous ZBP1 efficiently accumulated in nuclear foci, which overlapped PML oncogenic domains (PODs) or nuclear bodies (NBs). In transfection assays, the unique C-terminal region of ZBP1 was necessary for its typical cytoplasmic localization. Interestingly, the Zalpha-deleted form displayed an increased association with PODs compared to wild-type and, unlike wild-type, perfectly accumulated in PODs in LMB-treated cells, implying that the presence of Zalpha domain also facilitates the cytoplasmic localization. Our results demonstrate that ZBP1 is localized primarily in the cytoplasm but also associated with nuclear PODs in IFN or LMB-treated cells. Given that about half of ZBP1 mRNA lacks exon 2 encoding the Zalpha domain, our data also suggest that the localization of ZBP1 may be differentially regulated by the Z-DNA binding domain, Zalpha, in splice variants.

Animals↗
PubMed2006

Mouse LGI3 gene: expression in brain and promoter analysis.

Leucine-rich glioma inactivated 3 (LGI3) is a member of LGI/epitempin family of which the first member, LGI1/epitempin, was shown to be mutated in glioma and autosomal dominant lateral temporal epilepsy. Similar to LGI1, LGI3 is expressed predominantly in brain and its function is unknown. In this study, we examined the expression of mouse LGI3 (mLGI3) in adult and developing brain and analyzed the 5'-upstream transcriptional regulatory regions of mLGI3 gene. In situ hybridization showed that mLGI3 was expressed in widespread areas with selective regional variation in adult brain. In developing brain, mLGI3 mRNA was expressed at low level during embryo stages and markedly increased in broad areas after birth. Analysis of the 5'- and 3'-ends of mLGI3 mRNA identified a single transcription start site and two alternative 3'-ends. Luciferase reporter analysis using Neuro-2a cells and electrophoretic mobility shift assays identified a neuronal restrictive silencer element (NRSE; -2573 approximately -2553) and a phorbol ester-sensitive AP-2 element with repressor activity (-44 approximately -33) among multiple positive and negative regulatory regions. Since NRSE and AP-2 are implicated in neuron-specific gene expression and developmental regulation of many genes in brain, respectively, these results suggested that NRSE and AP-2 might play important roles in regulation of mLGI3 expression in brain.

Animals↗
PubMed2006

Biochemical characterization and preliminary X-ray crystallographic study of the domains of human ZBP1 bound to left-handed Z-DNA.

ZBP1 is involved in host responses against cellular stresses, including tumorigenesis and viral infection. Structurally, it harbors two copies of the Zalpha domain containing the Zalpha motif, at its N terminus. Here, we attempted to characterize the Z-DNA binding activities of two Zalpha domains in the human ZBP1, hZalpha(ZBP1) and hZbeta(ZBP1), using circular dichroism (CD). Our results indicated that both hZalpha(ZBP1) and hZbeta(ZBP1) are viable Z-DNA binders, and their binding activities are comparable to those of previously-established Zalpha domains. Additionally, we crystallized hZbeta(ZBP1) in a complex with Z-DNA, d(TCGCGCG)2. The crystal diffracted to 1.45 angstroms, and belongs to the P2(1)2(1)2(1) space group, with the unit-cell parameters: a = 29.53 angstroms, b = 58.25 angstroms, and c = 88.61 angstroms. The delineation of this structure will provide insight into the manner in which diverse Zalpha motifs recognize Z-DNA.

Amino Acid Motifs↗
PubMed2005

In-plane enyne metathesis and subsequent Diels-Alder reactions on self-assembled monolayers.

We report in-plane enyne metathesis and subsequent Diels-Alder reactions on self-assembled monolayers (SAMs) terminating in vinyl and acetylenyl groups on gold. After the formation of SAMs of vinyl and acetylenyl group-containing dithiols on gold, in-plane enyne metathesis of the vinyl and acetylenyl groups, leading to the formation of 1,3-diene, was achieved on the SAMs, and Diels-Alder reactions were then successfully performed with tetracyanoethylene, maleic anhydride, and maleimide. The reactions were confirmed by FT-IR spectroscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary-ion mass spectrometry. In-plane enyne metathesis developed herein would offer a versatile platform for the functionalization of surfaces with mild reaction conditions and a high compatibility in functional groups.

Letter↗
PubMed2005

Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases.

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B-Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B-Z junction as revealed by X-ray crystallography at 2.6 A resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification.

Base Pairing↗
PubMed2005

Surface-initiated growth of poly d(A-T) by Taq DNA polymerase.

In this paper, we report surface-initiated d(A-T) polymerization by Taq DNA polymerase as a method for constructing DNA-tethered surfaces using an enzyme. The enzymatic polymerization was conducted successfully via two steps: tethering of oligo d(A-T)s onto the surface presenting carboxylic acids by amide coupling and surface-initiated polymerization using Taq DNA polymerase. In this enzymatic polymerization process, the design and construction of carboxylic acid-presenting surfaces were found to be an important factor: DNA growth did not occur on the gold surface coated only with the self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA), but effectively proceeded on the surfaces presenting mixed SAMs of MHDA and 1-pentadecanethiol. The coupling of oligo d(A-T)s and the subsequent DNA polymerization reaction were characterized by polarized infrared external reflectance spectroscopy, ellipsometry, X-ray photoelectron spectroscopy, and atomic force microscopy.

Gold↗
PubMed2005

The target of RNAIII-activating protein (TRAP) from Staphylococcus aureus: purification, crystallization and preliminary X-ray analysis.

The target of the RNAIII-activating protein (TRAP) is a 21 kDa protein in which phosphorylation is activated by the RNAIII-activating protein (RAP), which causes an increase in RNAII and RNAIII synthesis and the production of the virulence factors. In an attempt to examine the structural role of TRAP in the signal transduction pathway, TRAP from Staphylococcus aureus was overexpressed, purified and crystallized using PEG 8000 and 5% Jeffamine M600 (pH 7.0), as precipitants by hanging-drop vapour diffusion methods at 287 K. The crystals belong to the orthorhombic space group, P2(1)2(1)2(1), with unit cell parameters of a=39.68, b=50.41, c=85.45 A. There is one monomer of TRAP per crystallographic asymmetric unit with a crystal volume per protein mass (V(M)) of 2.06 A(3) Da(-1) and a solvent content of 40.3%. A complete data set diffracting to 1.9 A resolution was collected from a single crystal at 100 K using a synchrotron-radiation source.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Distinct pH modulation for dual function of Galphah (transglutaminase II).

Galpha(h), also known as transglutaminase II, has GTPase as well as transglutaminase activities. To better understand the factors affecting these dual enzymatic activities, we examined the optimal pH (at 25 degrees C) and thermal stability (at 37 degrees C) of the activities using membranous Galpha(h) from mouse heart. The optimum pH for the GTPase activity of Galpha(h) is approximately 7.0. As well, the GTP binding activity of Galpha(h) is more thermostable at pH 7.0 than that at pH 9.0. Consistent with these observations on the GTPase function of Galpha(h), both the phospholipase C-delta1 activity and the yield of co-immunoprecipitation of Galpha(h)-coupled phospholipase C-delta1 in alpha(1)-adrenoceptor/Galpha(h)/phospholipase C-delta1 complex preparations were enhanced by incubation with an alpha(1)-agonist, phenylephrine, at pH 7.0. On the other hand, the transglutaminase activity of Galpha(h) is higher in the basic pH range with an optimum activity at pH approximately 9.0. Also, the transglutaminase activity of Galpha(h) is more thermostable at pH 9.0 than that at pH 7.0. These results indicate not only pH as a modulator for the dual functions of Galpha(h), but also provide direct evidence for the involvement of pH in the Galpha(h)-mediated alpha(1)-adrenoceptor signaling system in vitro.

Animals↗
PubMed2005

Crystallization and preliminary X-ray crystallographic study of the viral Zalpha domain bound to left-handed Z-DNA.

The Zalpha domain (yabaZalpha(E3L)) of the E3L protein homologue from Yaba-like disease virus, a yatavirus, was co-crystallized with d(TCGCGCG)(2) in the Z-conformation. The crystals belong to the P2(1)2(1)2 space group, with unit-cell parameters a=51.20 Angstroms, b=92.45 Angstroms, c=48.02 Angstroms, alpha=beta= gamma=90 degrees. The diffraction data were collected up to a resolution of 2.2 Angstroms. The structure of viral Zalpha motif will provide an insight into how diverse Zalpha motifs recognize Z-DNA.

Crystallization↗
PubMed2004

A poxvirus protein forms a complex with left-handed Z-DNA: crystal structure of a Yatapoxvirus Zalpha bound to DNA.

A conserved feature of poxviruses is a protein, well characterized as E3L in vaccinia virus, that confers IFN resistance on the virus. This protein comprises two domains, an N-terminal Z-DNA-binding protein domain (Zalpha) and a C-terminal double-stranded RNA-binding domain. Both are required for pathogenicity of vaccinia virus in mice infected by intracranial injection. Here, we describe the crystal structure of the Zalpha domain from the E3L-like protein of Yaba-like disease virus, a Yatapoxvirus, in a complex with Z-DNA, solved at a 2.0-A resolution. The DNA contacting surface of Yaba-like disease virus Zalpha(E3L) closely resembles that of other structurally defined members of the Zalpha family, although some variability exists in the beta-hairpin region. In contrast to the Z-DNA-contacting surface, the nonbinding surface of members of the Zalpha family are unrelated; this surface may effect protein-specific interactions. The presence of the conserved and tailored Z-DNA-binding surface, which interacts specifically with the zigzag backbone and syn base diagnostic of the Z-form, reinforces the importance to poxvirus infection of the ability of this protein to recognize the Z-conformation.

Amino Acid Sequence↗
PubMed2004

The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro.

The N-terminal domain of the vaccinia virus protein E3L (Z alpha(E3L)) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). Here, we report the solution structure of Z alpha(E3L) and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Z alpha(E3L) are very similar to the high-affinity Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). However, the key Z-DNA contacting residue Y48 of Z alpha(E3L) adopts a different side chain conformation in unbound Z alpha(E3L), which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower in vitro affinity of Z alpha(E3L) to Z-DNA compared with its homologues.

Amino Acid Sequence↗
PubMed2004

Evidence that vaccinia virulence factor E3L binds to Z-DNA in vivo: Implications for development of a therapy for poxvirus infection.

The E3L gene product found in all poxviruses is required for the lethality of mice in vaccinia virus infection. Both the C-terminal region, consisting of a double-stranded RNA-binding motif, and the N-terminal region (vZ(E3L)), which is similar to the Zalpha family of Z-DNA-binding proteins, are required for infection. It has recently been demonstrated that the function of the N-terminal domain depends on its ability to bind Z-DNA; Z-DNA-binding domains from unrelated mammalian proteins fully complement an N-terminal deletion of E3L. Mutations that decrease affinity for Z-DNA have similar effects in decreasing pathogenicity. Compounds that block the Z-DNA-binding activity of E3L may also limit infection by the poxvirus. Here we show both an in vitro and an in vivo assay with the potential to be used in screening for such compounds. Using a conformation-specific yeast one-hybrid assay, we compared the results for Z-DNA binding of vZ(E3L) with those for human Zbeta(ADAR1), a peptide that has similarity to the Zalpha motif but does not bind Z-DNA, and with a mutant of hZbeta(ADAR1), which binds Z-DNA. The results suggest that this system can be used for high-throughput screening.

Amino Acid Sequence↗
PubMed2004

Inhibition of nitric oxide generation by 23,24-dihydrocucurbitacin D in mouse peritoneal macrophages.

Nitric oxide (NO) has various physiological functions. However, uncontrolled overproduction of NO can be toxic in many pathologic conditions involving inflammatory tissue damage. In the present study, we examined effects of 23,24-dihydrocucurbitacin D (DHCD) isolated from the root of Bryonia alba L. on macrophage NO generation. DHCD (<80 microM) effectively abolished NO generation from macrophages activated with lipopolysaccharide and interferon-gamma. DHCD decreased the levels of protein and mRNA for inducible NO synthase (iNOS). DHCD potently blocked nuclear factor-kappaB (NF-kappaB) activation, a process necessary for transcriptional activation of iNOS. These results suggested that DHCD inhibited NO generation by blocking NF-kappaB activation and iNOS gene transcription. Because NF-kappaB activation is necessary not only for NO generation but also for many inflammatory processes, DHCD and its derivatives could be developed as anti-inflammatory drugs.

Animals↗
PubMed2003

Ca2+: a stabilizing component of the transglutaminase activity of Galphah (transglutaminase II).

Galphah (transglutaminase type II; tissue transglutaminase) is a bifunctional enzyme with transglutaminase (TGase) and guanosine triphosphatase (GTPase) activities. The GTPase function of Galphah is involved in hormonal signaling and cell growth while the TGase function plays an important role in apoptosis and in cross-linking extracellular and intracellular proteins. To analyze the regulation of these dual enzymatic activities we examined their calcium-dependence and thermal stability in enzymes from several cardiac sources (mouse heart, and normal, ischemic and dilated cardiomyopathic human hearts). The GTP binding activity of Galphah was markedly inhibited by Ca2+ whereas the TGase activity was strongly stimulated, suggesting that Ca2+ acts as a regulator, switching Galphah from a GTPase to a TGase. The TGase function of Galphah of both mouse and human hearts was more thermostable in the presence of Ca2+.

Animals↗
PubMed2003

A role for Z-DNA binding in vaccinia virus pathogenesis.

The N-terminal domain of the E3L protein of vaccinia virus has sequence similarity to a family of Z-DNA binding proteins of defined three-dimensional structure and it is necessary for pathogenicity in mice. When other Z-DNA-binding domains are substituted for the similar E3L domain, the virus retains its lethality after intracranial inoculation. Mutations decreasing Z-DNA binding in the chimera correlate with decreases in viral pathogenicity, as do analogous mutations in wild-type E3L. A chimeric virus incorporating a related protein that does not bind Z-DNA is not pathogenic, but a mutation that creates Z-DNA binding makes a lethal virus. The ability to bind the Z conformation is thus essential to E3L activity. This finding may allow the design of a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L.

Adenosine Deaminase↗