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Shigeo Murata

Publications and source records attributed to Shigeo Murata.

31 records · Page 2Linked to original sources
PubMed2004

Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor.

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.

Animals↗
PubMed2004

Formalin-fixed tumor cells effectively induce antitumor immunity both in prophylactic and therapeutic conditions.

BACKGROUND: Autologous whole tumor cell-based vaccinations would seem to be ideal since such vaccinations, in contrast to vaccination with a single defined antigen, have the potential to elicit a broad type of T-cell immune response to tumor-associated antigens. OBJECTIVE: We modified formaldehyde (formalin)-fixed mouse melanoma cells and investigated the utility of those cells as sources of tumor antigens for immunotherapy. METHODS: C57BL/6 or the proteasome activator PA28alpha-knockout mice were intradermally inoculated with 1% formalin-fixed B16 cells three times at weekly intervals either before or after tumor challenge. Simultaneously, interleukin-12 gene was transferred into the skin around immunization sites using gene gun technology. The effects were evaluated by tumor growth, antigen-specific interferon-gamma production in splenic lymphocytes, and activation of dendritic cells. RESULTS: Fixed cells directly induced production of tumor necrosis factor-alpha in dendritic cells more effectively than did frozen and thawed cells. More than 60% of the mice immunized with fixed cells and interleukin-12 rejected the challenged B16 tumor. CD4+ T cells from those mice produced a significant amount of interferon-gamma in response to melanoma cells. Furthermore, this combined treatment showed antitumor immunity initiated by CD8+ and CD4+ T cells in the therapeutic experiments. PA28alpha/beta appeared not to be required for the development of CD8+ T cells, although it is known to be essential for the development of CD8+ T cells specific for tyrosinase-related protein-2, one of melanocyte-lineage differentiated antigens. CONCLUSION: These results suggest that formalin-fixed autologous melanoma cells have a potential to function as effective antigen sources for immunotherapy.

Animals↗
PubMed2004

Structural basis for distinct roles of Lys63- and Lys48-linked polyubiquitin chains.

Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves as a signalling function that controls a wide variety of cellular processes. To date, two major forms of polyubiquitin chain have been functionally characterized, in which the isopeptide bond linkages involve Lys48 or Lys63. Lys48-linked polyubiquitin tagging is mostly used to target proteins for degradation by the proteasome, whereas Lys63-linked polyubiquitination has been linked to numerous cellular events that do not rely on degradative signalling via the proteasome. Apparently linkage-specific conformations of polyubiquitin chains are important for these cellular functions, but the structural bases distinguishing Lys48- and Lys63-linked chains remain elusive. Here, we report NMR and small-angle X-ray scattering (SAXS) studies on the intersubunit interfaces and conformations of Lys63- and Lys48-linked di- and tetraubiquitin chains. Our results indicate that, in marked contrast to Lys48-linked chains, Lys63-linked chains are elongated molecules with no stable non-covalent intersubunit interfaces and thus adopt a radically different conformation from that of Lys48-linked chains.

Lysine↗
PubMed2004

Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA vaccination with a chimeric gene encoding a syngeneic cytotoxic T lymphocyte epitope of melanocyte and green fluorescent protein.

Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8(+) cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2(181-188)) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2(181-188). GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28alpha/beta. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2(181-188), by activating CD8(+) CTL specific for TRP-2(181-188). TRP-2(181-188) fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8(+) T cells specific for the CTL epitope. Furthermore, CD4(+) T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the "ubiquitin-proteasome pathway". It is noteworthy to document that genetic immunization with pGFP plus pTRP-2(181-188) failed to exert the antitumour immunity.

Animals↗
PubMed2003

Sterol regulatory element-binding proteins are negatively regulated through SUMO-1 modification independent of the ubiquitin/26 S proteasome pathway.

Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that activate the genes involved in cholesterol and fatty acid biosynthesis. We here report that the nuclear forms of SREBPs are modified by the small ubiquitin-related modifier (SUMO)-1. Mutational analyses identified two major sumoylation sites (Lys(123) and Lys(418)) in SREBP-1a and a single site (Lys(464)) in SREBP-2. Mutant SREBPs lacking one or two sumoylation sites exhibited increased transactivation capacity on an SREBP-responsive promoter. Overexpression of SUMO-1 reduced whereas its dominant negative form increased mRNA levels of SREBP-responsive genes. Nuclear SREBPs interacted with the SUMO-1-conjugating enzyme Ubc9, and overexpression of a dominant negative form of Ubc9 increased the mRNA levels of SREBP-responsive genes. Pulse-chase experiments revealed that sumoylation did not affect the degradation of SREBPs through the ubiquitin-proteasome pathway. In vitro ubiquitylation assay showed no competition between ubiquitin and SUMO-1 for the same lysine. Considered together, our results indicate that SUMO-1 modification suppresses the transactivation capacity of nuclear SREBPs in a manner different from the negative regulatory mechanism mediated by proteolysis.

Animals↗
PubMed2003

CHIP: a quality-control E3 ligase collaborating with molecular chaperones.

It is notable that both the chaperone and ubiquitin-proteasome systems are required for removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. Carboxyl-terminus of Hsc70 interacting protein (CHIP), originally identified as a co-chaperone of Hsc70, has both a tetratricopeptide repeat (TPR) motif and a U-box domain. The TPR motif associates with Hsc70 and Hsp90, while the U-box domain executes a ubiquitin ligase activity. Thus, CHIP is an ideal molecule acting as a protein quality-control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. Accumulating evidence from in vitro studies indicates that this is apparently the case. Here, we present and discuss several unresolved but critical issues related to the molecular mechanism and in vivo roles of CHIP.

Animals↗
PubMed2003

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.

Active Transport, Cell Nucleus↗
PubMed2003

[The protein quality control and neurodegeneration].

There are growing lines of evidence addressing a potential link between failure of protein quality control and neurodegeneration. Hence, it is important to know the quality control mechanism of the cell for understanding the molecular basis underlying neurodegenerative diseases. To date, it is notable that molecular chaperones and the ubiquitin-proteasome system play a central role for maintenance of protein homeostasis by catalyzing refolding and/or the immediate destruction of misfolded or impaired proteins generated in cells. However, how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains largely elusive. In this article, we discuss the mechanism of neurodegeneration, based on the protein quality control mediated by the ubiquitin-proteasome system in the cell.

Cysteine Endopeptidases↗
PubMed2002

Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity.

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cerebral cortex, brainstem, and spinal cord. The cytopathological hallmark in the remaining motor neurons of ALS is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. In this paper we report that Dorfin, a RING finger-type E3 ubiquitin ligase, is predominantly localized in the inclusion bodies of familial ALS with a copper/zinc superoxide dismutase (SOD1) mutation as well as sporadic ALS. Dorfin physically bound and ubiquitylated various SOD1 mutants derived from familial ALS patients and enhanced their degradation, but it had no effect on the stability of the wild-type SOD1. The overexpression of Dorfin protected against the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions. Our results indicate that Dorfin protects neurons by recognizing and then ubiquitylating mutant SOD1 proteins followed by targeting them for proteasomal degradation.

Aged↗
PubMed2002

Two distinct pathways mediated by PA28 and hsp90 in major histocompatibility complex class I antigen processing.

Major histocompatibility complex (MHC) class I ligands are mainly produced by the proteasome. Herein, we show that the processing of antigens is regulated by two distinct pathways, one requiring PA28 and the other hsp90. Both hsp90 and PA28 enhanced the antigen processing of ovalbumin (OVA). Geldanamycin, an inhibitor of hsp90, almost completely suppressed OVA antigen presentation in PA28alpha(-/-)/beta(-/-) lipopolysaccharide blasts, but not in wild-type cells, indicating that hsp90 compensates for the loss of PA28 and is essential in the PA28-independent pathway. In contrast, treatment of cells with interferon (IFN)-gamma, which induces PA28 expression, abrogated the requirement of hsp90, suggesting that IFN-gamma enhances the PA28-dependent pathway, whereas it diminishes hsp90-dependent pathway. Importantly, IFN-gamma did not induce MHC class I expressions in PA28-deficient cells, indicating a prominent role for PA28 in IFN-gamma-stimulated peptide supply. Thus, these two pathways operate either redundantly or specifically, depending on antigen species and cell type.

Amino Acid Sequence↗
PubMedDate not supplied

Multiple chaperone-assisted formation of mammalian 20S proteasomes.

Protein degradation is essential for maintenance of cellular homeostasis. The majority of proteins are selectively degraded in eukaryotic cells by the ubiquitin-proteasome system. The 26S proteasome selects target proteins that are covalently modified with polyubiquitin chains. The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. The catalytic activities are carried out by the core 20S proteasome. The eukaryotic 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7. Recent studies have revealed the mechanism responsible for the assembly of such a complex structure. This article recounts the observations that disclosed the biogenesis of 20S proteasomes and discusses the difference in the mechanism of assembly between archael, yeast, and mammalian 20S proteasomes.

Animals↗