Biomedical subjects

Kohsuke Kataoka

Publications and source records attributed to Kohsuke Kataoka.

10 recordsLinked to original sources

PubMed2006

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles.

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.

Animals↗

PubMed2005

Synergistic activation of the insulin gene promoter by the beta-cell enriched transcription factors MafA, Beta2, and Pdx1.

Specific expression of the insulin gene in pancreatic islet beta-cells requires multiple cis-regulatory elements in its promoter. Pdx1, MafA, and Beta2 have been identified as beta-cell enriched transcription factors that bind to these elements. Pdx1 has been shown to bind to A1, A3, A5, and GG2, and Beta2 binds to E1 by forming a heterodimer with the ubiquitous factor E47. MafA was recently identified as a C1-element binding factor. However, interactions between these factors and the promoter have not been characterized in detail. In this report, we show that these transactivators synergistically stimulate insulin promoter activity. Among multiple binding sites for Pdx1, MafA, and Beta2, at least GG2, C1, and E1 elements located in the promoter region between -150 and -100 base pairs are necessary for the synergism. We also found that neither MafB nor c-Maf, close relatives of MafA, showed synergistic activation. These results suggest that co-expression and functional synergism of these beta-cell enriched transactivators, MafA, Pdx1, and Beta2, are critical for establishing the beta-cell-specific and efficient expression of the insulin gene.

Basic Helix-Loop-Helix Proteins↗

PubMed2005

Selective ligand purification using high-performance affinity beads.

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.

Animals↗

PubMed2004

Rep68 protein of adeno-associated virus type 2 interacts with 14-3-3 proteins depending on phosphorylation at serine 535.

Rep78/68 proteins of adeno-associated virus type 2 (AAV-2) are involved in many aspects of the viral life cycle, including replication, gene expression, and site-specific integration. To understand the molecular mechanisms of the actions of Rep proteins, we searched for Rep68-interacting cellular proteins by utilizing a one-step affinity purification technique and identified two members of 14-3-3 proteins (14-3-3 epsilon and gamma). We found that phosphorylation of 535Ser at the carboxy terminus of Rep68 was critical for its association with 14-3-3. The association of 14-3-3 proteins to Rep68 resulted in reduction of the affinity of Rep68 for DNA. Furthermore, genome DNA replication of a recombinant mutant virus carrying a phosphorylation-deficient Rep68 (Ser535Ala) was more efficient than that of the wild-type virus. These results suggest that phosphorylation of Rep68 and subsequent association with 14-3-3 proteins regulates Rep-mediated functions during the AAV life cycle.

14-3-3 Proteins↗

PubMed2003

MafA has strong cell transforming ability but is a weak transactivator.

The maf oncogene of the avian oncogenic retrovirus AS42 encodes a nuclear bZip protein, v-Maf, that recognizes sequences related to the AP-1 target site. The corresponding cellular protein, c-Maf belongs to a family of related bZip proteins together with MafA and MafB. In this paper, we compare the transactivation and cell transforming abilities of MafA and MafB along with two forms of the c-Maf protein. These proteins induce cellular transformation when expressed in chicken embryo fibroblasts. In reporter assays, MafA is a much less effective transactivator than the other Maf proteins, but unexpectedly shows the strongest activity in cell transformation. Chimeras of MafA and MafB correlate the strong cell transforming ability of MafA with its DNA-binding domain. The DNA-binding domain of MafA is also correlated with weak transactivation. Additional mutagenesis experiments show that transactivation and transformation by MafA are also controlled by phosphorylation of two conserved serine residues in the transactivation domain. Finally, we constructed MafA-estrogen receptor fusion molecules that show tightly hormone-dependent cell transforming ability. These regulatable constructs permit a kinetic characterization of target gene responses and facilitate discrimination between direct and indirect targets.

Amino Acid Sequence↗

PubMed2003

Artificial oncoproteins: modified versions of the yeast bZip protein GCN4 induce cellular transformation.

We have constructed artificial AP-1 proteins containing elements derived from yeast GCN4 and from the herpes simplex virus activator VP16. These proteins can only homodimerize but do not heterodimerize, and lacking significant homology to Jun outside the DNA-binding domain, they are largely unaffected by proteins that modulate Jun. Constructs in which the transactivation domain of GCN4 is replaced by that of VP16 induce oncogenic transformation in cultures of chicken embryo fibroblasts. The availability of transforming VP16-GCN4 fusion proteins permits an evaluation of downstream target genes, based on the hypothesis that transformation-relevant targets should be common between Jun and the artificial AP-1 proteins. In a pilot study, we examined the expression of several Jun target genes in cells transformed by the VP16-GCN4 fusions and found that some of the Jun targets are not upregulated by the GCN4-derived transforming construct, suggesting that their upregulation in Jun-transformed cells is not essential for cell transformation. We have further constructed a regulatable GCN4-VP16 protein that will permit a kinetic characterization of target gene responses and will facilitate discrimination between direct and indirect targets.

Animals↗

PubMed2003

Simian virus 40 VP1 capsid protein forms polymorphic assemblies in vitro.

The simian virus 40 (SV40) capsid is composed of 72 pentamers of VP1, the major protein of SV40. These pentamers are arranged in a T=7d icosahedral surface lattice, which is maintained by three types of appropriately arranged, non-equivalent interactions between the pentamers. However, it remains unclear how these interactions are achieved. In this study, the in vitro assembly of recombinant VP1 was analysed. Electron microscopy observations revealed that these recombinant VP1 proteins assembled into structurally polymorphic particles depending on environmental conditions. VP1 pentamers assembled efficiently into virus-like particles (VLPs) when high concentrations of ammonium sulfate were present. However, in the presence of 1 M NaCl and 2 mM CaCl(2) at neutral pH, VP1 pentamers formed not only VLPs but also produced tiny T=1 icosahedral particles and tubular structures. The exclusion of CaCl(2) resulted in the exclusive formation of tiny particles. In contrast, in the presence of 150 mM NaCl at pH 5, the VP1 pentamers produced only extraordinarily long tubular structures. VP1 is thus quite unique in that it can assemble into such diverse structures. These observations provide clues that will help elucidate the mechanisms underlying SV40 capsid formation.

Animals↗

PubMed2002

MafA is a glucose-regulated and pancreatic beta-cell-specific transcriptional activator for the insulin gene.

The insulin gene is specifically expressed in beta-cells of the Langerhans islets of the pancreas, and its transcription is regulated by the circulating glucose level. Previous reports have shown that an unidentified beta-cell-specific nuclear factor binds to a conserved cis-regulatory element called RIPE3b and is critical for its glucose-regulated expression. Based on the sequence similarity of the RIPE3b element and the consensus binding sequence of the Maf family of basic leucine zipper transcription factors, we here identified mammalian homologue of avian MafA/L-Maf, an eye-specific member of the Maf family, as the RIPE3b-binding transcriptional activator. Reverse transcription-PCR analysis showed that mafA mRNA is detected only in the eyes and in pancreatic beta-cells and not in alpha-cells. MafA protein as well as its mRNA is up-regulated by glucose, consistent with the glucose-regulated binding of MafA to the RIPE3b element in beta-cell nuclear extracts. In transient luciferase assays, we also showed that expression of MafA greatly enhanced insulin promoter activity and that a dominant-negative form of MafA inhibited it. Therefore, MafA is a beta-cell-specific and glucose-regulated transcriptional activator for insulin gene expression and thus may be involved in the function and development of beta-cells as well as in the pathogenesis of diabetes.

Amino Acid Sequence↗

PubMed2002

Spatial redox regulation of a critical cysteine residue of NF-kappa B in vivo.

Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-kappaB. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiol-modifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-kappaB is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-kappaB. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-kappaB activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins.

Cysteine↗

PubMed2002

YEAF1/RYBP and YAF-2 are functionally distinct members of a cofactor family for the YY1 and E4TF1/hGABP transcription factors.

The transcription factor hGABP/E4TF1 is a heterotetrameric complex composed of two DNA-binding subunits (hGABP alpha/E4TF1-60) and two transactivating subunits (hGABP beta/E4TF1-53). In order to understand the molecular mechanism of transcriptional regulation by hGABP, we searched for proteins that interact with the non-DNA-binding subunit, hGABP beta, using yeast two-hybrid screening. We identified a human cDNA encoding a protein related to YAF-2 (YY1-associated factor 2), which was previously isolated as an interacting partner of the Ying-Yang-1 (YY1) transcription factor. Reflecting this similarity, both YAF-2 and this novel protein (named YEAF1 for YY1- and E4TF1/hGABP-associated factor-1) interacted with hGABP beta and YY1 in vitro and in vivo, indicating that YEAF1 and YAF-2 constitute a cofactor family for these two structurally distinct transcription factors. By using yeast three-hybrid assay, we demonstrated that hGABP beta and YY1 formed a complex only in the presence of YEAF1, indicating that YEAF1 is a bridging factor of these two transcription factors. These cofactors are functionally different in that YAF-2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity. Also, YAF-2 mRNA is highly expressed in skeletal muscle, whereas YEAF1 mRNA is highly expressed in placenta. We speculate that the transcriptional activity of hGABP is in part regulated by the expression levels of these tissue-specific cofactors. These results provide a novel mechanism of transcriptional regulation by functionally distinct cofactor family members.

Amino Acid Sequence↗