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Yuko Hirano

Publications and source records attributed to Yuko Hirano.

9 recordsLinked to original sources
PubMed2006

Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes.

The 20S proteasome is a catalytic core of the 26S proteasome, a central enzyme in the degradation of ubiquitin-conjugated proteins. It is composed of 14 distinct gene products that form four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). It is reported that the biogenesis of mammalian 20S proteasomes is assisted by proteasome-specific chaperones, named PAC1, PAC2, and hUmp1, but the details are still unknown. Here, we report the identification of a chaperone, designated PAC3, as a component of alpha rings. Although it can intrinsically bind directly to both alpha and beta subunits, PAC3 dissociates before the formation of half-proteasomes, a process coupled with the recruitment of beta subunits and hUmp1. Knockdown of PAC3 impaired alpha ring formation. Further, PAC1/2/3 triple knockdown resulted in the accumulation of disorganized half-proteasomes that are incompetent for dimerization. Our results describe a cooperative system of multiple chaperones involved in the correct assembly of mammalian 20S proteasomes.

Cell Line↗
PubMed2006

A new function for p53 ubiquitination.

The amount of p53 protein in a cell is normally limited by ubiquitin-dependent degradation. In this issue of Cell, Le Cam et al. (2006) reveal that p53 ubiquitination contributes to transcriptional activation rather than protein stability. These results may provide insight into how p53 can modulate diverse cellular processes such as growth arrest and apoptosis.

Cell Cycle Proteins↗
PubMed2005

A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes.

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7 (refs 4, 5), but the mechanism responsible for the assembly of such a complex structure remains elusive. Here we report two chaperones, designated proteasome assembling chaperone-1 (PAC1) and PAC2, that are involved in the maturation of mammalian 20S proteasomes. PAC1 and PAC2 associate as heterodimers with proteasome precursors and are degraded after formation of the 20S proteasome is completed. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes, whereas knockdown by short interfering RNA impairs it, resulting in poor maturation of 20S proteasomes. Furthermore, the PAC complex provides a scaffold for alpha-ring formation and keeps the alpha-rings competent for the subsequent formation of half-proteasomes. Thus, our results identify a mechanism for the correct assembly of 20S proteasomes.

Animals↗
PubMed2005

Large- and small-scale purification of mammalian 26S proteasomes.

The 26S proteasome is an ATP-dependent protease known to collaborate with ubiquitin, whose polymerization acts as a marker for regulated and enforced destruction of unnecessary proteins in eukaryotic cells. It is an unusually large multi-subunit protein complex, consisting of a central catalytic machine (called the 20S proteasome or CP/core particle) and two terminal regulatory subcomplexes, termed PA700 or RP/regulatory particle, that are attached to both ends of the central portion in opposite orientations to form an enzymatically active proteasome. To date, proteolysis driven by the ubiquitin-proteasome system has been shown to be involved in a diverse array of biologically important processes, such as the cell cycle, immune response, signaling cascades, and developmental programs; and the field continues to expand rapidly. Whereas the proteasome complex has been highly conserved during evolution because of its fundamental roles in cells, it has also acquired considerable diversity in multicellular organisms, particularly in mammals, such as immunoproteasomes, PA28, S5b, and various alternative splicing forms of S5a (Rpm 10). However, the details of the ultimate pathophysiological roles of mammalian proteasomes have remained elusive. This article focuses on methods for assay and purification of 26S proteasomes from mammalian cells and tissues.

Animals↗
PubMed2005

Erythropoietin-resistant anaemia in a predialysis patient with Klinefelter syndrome.

A diabetic predialysis patient who had significantly reduced sensitivity to erythropoietin therapy was admitted to Tsukuba University Hospital. Many factors that might have been the cause of the erythropoietin resistance were examined, and a diagnosis of refractory anaemia was made based on a bone marrow aspiration biopsy. A cytogenetic abnormality (47, XXY) was also detected in the bone marrow biopsy specimen, and hence the patient was also diagnosed with Klinefelter syndrome. It was suspected that the sex chromosome abnormality influenced glucose intolerance, renal insufficiency, and erythropoietin resistance due to myelodysplastic changes in the bone marrow.

Anemia↗
PubMed2003

Sterol regulatory element-binding protein-2 interacts with hepatocyte nuclear factor-4 to enhance sterol isomerase gene expression in hepatocytes.

In the course of an effort to identify unknown targets genes for sterol regulatory element-binding proteins (SREBPs) by PCR, the gene for ATP citrate-lyase was determined to be one such gene. (Sato, R., Okamoto, A., Inoue, J., Miyamoto, W., Sakai, Y., Emoto, N., Shimano, H., and Maeda, M. (2000) J. Biol. Chem. 275, 12497-12502). We here report that gene expression of sterol Delta8-isomerase (SI), which catalyzes the conversion of the 8-ene isomer into the 7-ene isomer in the last steps of the cholesterol biosynthetic pathway, is regulated by SREBPs, mainly by SREBP-2. Luciferase assays using the promoter of the human SI gene revealed that a 200-base pair segment upstream region from the transcription start site contains functional elements required for the activity of the SREBPs, Sp1 and NF-Y. Interestingly, SI gene expression was well regulated by sterols in Caco-2 and HepG2 cells, in contrast with HEK293 and HeLa cells. Overexpression of hepatocyte nuclear factor (HNF)-4 in HEK293 cells augmented expression of SREBP-responsive genes including the SI gene, whereas inactivation of HNF-4 by small interfering RNAs in HepG2 cells reduced the SI gene promoter activity. The in vitro pull-down and in vivo co-immunoprecipitation experiments showed the direct interaction between SREBP-2 and HNF-4. These data provide a novel pathway by which HNF-4 potentiates the SREBP functions and stimulates expression of SREBP-responsive genes in enterohepatic cells.

Animals↗
PubMed2003

A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6.

Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6alpha and ATF6beta), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355-1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6alpha and ATF6beta, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6alpha and ATF6beta by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.

Activating Transcription Factor 6↗
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↗