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Seishi Murakami

Publications and source records attributed to Seishi Murakami.

At least 19 recordsLinked to original sources
PubMed2006

Temperature-sensitive defects of the GSP1gene, yeast Ran homologue, activate the Tel1-dependent pathway.

RanGTPase is involved in many cellular processes. It functions in nuclear-cytosolic transport and centrosome formation. Ran also localizes to chromatin as RCC1 does, its guanine nucleotide exchange factor, but Ran's function on chromatin is not known. We found that gsp1, a temperature-sensitive mutant of GSP1, a Saccharomyces cerevisiae Ran homologue, suppressed the hydroxyurea (HU) and ultra violet (UV) sensitivities of the mec1 mutant. In UV-irradiated mec1 gsp1 cells, Rad53 was phosphorylated despite the lack of Mec1. This suppression depended on the TEL1 gene, given that the triple mutant, mec1 gsp1 tel1, was unable to grow. The gsp1 mutations also suppressed the HU sensitivity of the rad9 mutant in a Tel1-dependent manner, but not the HU sensitivity of the rad53 mutant. These results indicated that Rad53 was activated by the Tel1 pathway in mec1 gsp1 cells, suggesting that Gsp1 helps regulate the role switching the ATM family kinases Mec1 and Tel1.

Cell Proliferation↗
PubMed2006

Molecular functions and biological roles of hepatitis B virus x protein.

Chronic infection of hepatitis B virus (HBV) is one of the major causes of hepatocellular carcinoma (HCC) in the world. Hepatitis B virus X protein (HBx) has been long suspected to be involved in hepatocarcinogenesis, although its oncogenic role remains controversial. HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progress, protein degradation pathways, apoptosis, and genetic stability by directly or indirectly interacting with host factors. This review focuses on the biological roles of HBx in HBV replication and cellular transformation in terms of the molecular functions of HBx. Using the transient HBV replication assay, ectopically expressed HBx could stimulate HBV transcription and replication with the X-defective replicon to the level of those with the wild one. The transcription coactivation is mainly contributing to the stimulatory role of HBx on HBV replication although the other functions may affect HBV replication. Effect of HBx on cellular transformation remains controversial and was never addressed with human primary or immortal cells. Using the human immortalized primary cells, HBx was found to retain the ability to overcome active oncogene RAS-induced senescence that requires full-length HBx. At least two functions of HBx, the coactivation function and the ability to overcome oncogene-induced senescence, may be cooperatively involved in HBV-related hepatocarcinogenesis.

Amino Acid Sequence↗
PubMed2006

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon.

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

Alanine↗
PubMed2005

The SIT4 gene, which encodes protein phosphatase 2A, is required for telomere function in Saccharomyces cerevisiae.

Life span and number of cell divisions in eukaryotes are limited. The accumulation of stress-associated damage due to ageing may cause irreversible cell cycle arrest, so-called "cellular senescence". Although many genes have been implicated in determining life span, regulatory systems that counteract age-related stress have not yet been clarified. We examined senescence during a stress of Saccharomyces cerevisiae strains carrying disruptions in protein phosphatase (PPase)-encoding genes in order to identify the system counteracting senescence. Among these strains, short telomeres were found in the sit4 disruptant that lacks one form of protein phosphatase 2A (PP2A). Silencing ability in the subtelomeric region was impaired and hyperphosphorylation of Sir3 was also observed in this mutant. The sit4 mutant was found to have altered nucleoli and a life span as short as an sgs1 mutant. These observations suggest that the PP2A pathway regulates life span in yeast.

Cell Cycle↗
PubMed2005

Mutational analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein.

RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5.

Amino Acid Sequence↗
PubMed2005

The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication.

The role and functional domain of hepatitis B virus (HBV) X protein (HBx) in regulating HBV transcription and replication were investigated with a transient transfection system in the human hepatoma cell line HepG2 using wild-type or HBx-minus HBV genome constructs and a series of deletion or mutation HBx expression plasmids. We show here that HBx has augmentation effects on HBV transcription and replication as a HBV mutant genome with defective X gene led to decreased levels of 3.5-kb HBV RNA and HBV replication intermediates and that these decreases can be restored by either transient ectopic expression of HBx or a stable HBx expression cell line. The C-terminal two-thirds (amino acids [aa] 51 to 154), which contain the transactivation domain, is required for this function of HBx; the N-terminal one-third (aa 1 to 50) is not required. Using the alanine scanning mutagenesis strategy, we demonstrated that the regions between aa 52 to 65 and 88 to 154 are important for the augmentation function of HBx in HBV replication. By the luciferase reporter gene analysis, we found that the transactivation and coactivation activities of HBx coincide well with its augmentation function in HBV transcription and replication. These results suggest that HBx has an important role in stimulating HBV transcription and replication and that the transcriptional transactivation function of HBx may be critical for its augmentation effect on HBV replication.

Amino Acid Sequence↗
PubMed2004

Nucleolin interacts with telomerase.

Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits which plays essential roles in maintaining telomeres in actively dividing cells. Recent work indicates that telomerase shuttles between subcellular compartments during assembly and in response to specific stimuli. In particular, telomerase colocalizes with nucleoli in normal human fibroblasts. Here, we show that nucleolin, a major nucleolar phosphoprotein, interacts with telomerase and alters its subcellular localization. Nucleolin binds the human telomerase reverse transcriptase subunit (hTERT) through interactions with its RNA binding domain 4 and carboxyl-terminal RGG domain, and this binding also involves the telomerase RNA subunit hTERC. The protein-protein interaction between nucleolin and hTERT is critical for the nucleolar localization of hTERT. These findings indicate that interaction of hTERT and nucleolin participates in the dynamic intracellular localization of telomerase complex.

Catalytic Domain↗
PubMed2004

Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture.

The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication. We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. (2002) Biochem. Biophys. Res. Commun. 293, 993-999). The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins. We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells. The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon. Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication. Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon. By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity. However, heat- and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold.

Amino Acid Sequence↗
PubMed2004

Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon.

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.

Amino Acid Sequence↗
PubMed2004

Subcellular localization of RPB5-mediating protein and its putative functional partner.

We previously identified a novel cellular protein, RPB5-mediating protein (RMP), that retains corepressor activity and functionally antagonizes transcriptional modulation via hepatitis B virus X protein. The subcellular localization of RMP was examined using green fluorescent protein-fused protein forms. We found that a nuclear localization signal (NLS) and a coiled-coil (CC) domain functioning as a cytoplasmic localization signal (CLS) are important for the subcellular localization of RMP. The CLS apparently acts dominantly, since RMP was mostly localized in the cytoplasm with weak and diffuse signals in the nucleus, and the NLS was indispensable for the nuclear localization of RMP only in the absence of the CLS. Using a yeast two-hybrid method, we isolated a putative corepressor, DNA methyltransferase 1-associating protein (DMAP1), which was found to bind to the CC domain of RMP. DMAP1 facilitated the nuclear localization of RMP and the corepressor activity of RMP in a dose-dependent manner by interacting with the CC domain of RMP. These results are discussed in light of a recent paper showing a novel evolutionarily conserved role of URI in the TOR signaling pathway.

Animals↗
PubMed2004

[Purification and partial characterization of hepatitis C virus (HCV) non-structural protein 5A (NS5A) expressed in Escherichia coli].

It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication. In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method. The native lysis buffer was used to remove most of the soluble non-specific proteins. His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin. The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein. The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt). The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro. Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.

DNA Primers↗
PubMed2003

Hepatitis B virus X protein induces angiogenesis by stabilizing hypoxia-inducible factor-1alpha.

Hepatitis B virus X protein (HBx) is closely involved in the development of hepatocellular carcinoma, a highly vascularized solid tumor. Here we show that HBx increases the transcriptional activity and protein level of hypoxia-inducible factor-1alpha (HIF-1alpha) under both normoxic and hypoxic conditions, and it also stimulates angiogenesis. HBx directly interacted with the bHLH/PAS domain of HIF-1alpha but not with the von Hippel-Lindau protein (pVHL). HBx decreased the binding of pVHL to HIF-1alpha and prevented ubiquitin-dependent degradation of HIF-1alpha. In HBx-transgenic mice, HIF-1alpha and vascular endothelial growth factor were strongly detected in the dysplastic lesion, where HBx was also more highly expressed than in the non-neoplastic region of the liver. An immunohistochemical study showed that microvessels are more abundant in the dysplastic lesion than in the non-neoplastic region. Our data suggest that HBx stabilizes HIF-1alpha and leads to angiogenesis during hepatocarcinogenesis.

Animals↗
PubMed2003

Telomerase maintains telomere structure in normal human cells.

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.

Antibodies, Monoclonal↗
PubMed2003

Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase.

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is a potentially useful diagnostic marker for cancers. There have been few studies in which immunological detection of hTERT has been attempted and its subcellular localization has not been precisely defined. In the present study, we re-evaluated expression and localization of hTERT in cancer and normal cells using a newly developed antibody. Immunohistochemistry revealed that hTERT is expressed in approximately 80% of gynecological cancers, but some premalignant lesions exhibited weak expression of hTERT. Interestingly, not only nuclei but also cytoplasm of cancer cells were positive for hTERT staining. This finding was supported by the results of Western blot analysis of cell lines, in which both nuclear and cytoplasmic extracts exhibited significant hTERT bands. Cytoplasmic hTERT in cancer cells may be functional because the telomeric repeat amplification protocol assay of cytoplasmic extracts showed high levels of telomerase activity. Unexpectedly, not all normal primary cells and telomerase-negative cancer cell lines lacked hTERT expression; some exhibited weak TERT signals. In Western analysis, hTERT signals did not always correlate with telomerase activity of the various cell types. These findings suggest that functional hTERT is expressed in both the nucleus and cytoplasm of cancer cells and that hTERT expression does not strictly reflect telomerase activity. Further analysis is needed to clarify the biological significance of cytoplasmic hTERT.

Blotting, Western↗
PubMed2003

Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP).

RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase II as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74 of TFIIF subunits. In the immunoprecipitation assay in COS1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-amino-acid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.

Animals↗
PubMed2002

Direct interaction between nucleolin and hepatitis C virus NS5B.

Hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme in HCV replication. While studying the subcellular localization of a NS5B mutant lacking the C-terminal membrane-anchoring domain, NS5Bt, we found that expression of the green fluorescent protein (GFP)-fused form was exclusively nucleolar. Interestingly, the distribution of endogenous nucleolin changed greatly in the cells expressing GFP-NS5B, with nucleolin colocalized with GFP-NS5B in perinuclear regions in addition to the nucleolus, suggesting that NS5B retains the ability to bind nucleolin. The interaction between nucleolin and NS5B was demonstrated by GST pull-down assay. GST pull-down assay results indicated that C-terminal region of nucleolin was important for its binding to NS5B. Scanning clustered alanine substitution mutants library of NS5B revealed two sites on NS5B that binds nucleolin. NS5B amino acids 208-214 and 500-506 were both found to be indispensable for the nucleolin binding. We reported that the latter sequence is essential for oligomerization of NS5B, which is a prerequisite for the RdRP activity. C-terminal nucleolin inhibited the NS5B RdRP activity in a dose-dependent manner. Taken together, this indicates the binding ability of nucleolin may be involved in NS5B functions.

Animals↗
PubMed2002

Identification of serum anti-human telomerase reverse transcriptase (hTERT) auto-antibodies during progression to hepatocellular carcinoma.

Human telomerase reverse transcriptase, hTERT, is the catalytic component of human telomerase. Expression of hTERT confers telomerase activity, indicating that hTERT is the rate-limiting component of human telomerase. Here we report the detection of anti-hTERT auto-antibodies in the sera derived patients with hepatocellular carcinoma using recombinant, purified hTERT as an antigen in an enzyme linked immunosorbent assay (ELISA). The levels of anti-hTERT antibodies in serum correlated with progression to hepatocellular carcinoma. In contrast, we detected only low levels of anti-hTERT auto-antibodies in the sera derived from 18 normal volunteers. The observation of hTERT auto-antibodies in the sera derived from cancer patients suggests that such auto-antibodies constitute novel and specific tumor marker.

Adult↗