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Nobuko Hosokawa

Publications and source records attributed to Nobuko Hosokawa.

13 recordsLinked to original sources
PubMed2006

EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming.

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Amino Acid Sequence↗
PubMed2006

EDEM accelerates ERAD by preventing aberrant dimer formation of misfolded alpha1-antitrypsin.

Misfolded glycoproteins are degraded by a mechanism known as ERAD (ER-associated degradation) after retrotranslocation out of the endoplasmic reticulum (ER). This mechanism plays an important role in ER quality control. We previously reported that an ER membrane protein, EDEM, accelerates ERAD of a misfolded alpha1-antitrypsin variant, null (Hong Kong) (NHK), suggesting that EDEM may function as an acceptor of terminally misfolded glycoproteins. In this study, we constructed several genetically manipulated cell lines to test this hypothesis. EDEM expression did not alter the secretion rate of properly folded molecules and the forced retention of wild-type alpha1-antitrypsin in the ER did not cause its association with EDEM, suggesting that EDEM may function as a molecular chaperone. To examine this possibility, we analyzed the effect of EDEM over-expression on the structure of NHK, and found that the accumulation of covalent NHK dimers was selectively prevented by the over-expression of EDEM. Co-expression of NHK with two other ER membrane proteins, calnexin and H(+)/K(+)-ATPase (beta subunit), did not inhibit NHK dimer formation or accelerate NHK ERAD. These results indicate that EDEM may maintain the retrotranslocation competence of NHK by inhibiting aggregation so that unstable misfolded proteins can be accommodated by the dislocon for ERAD.

Adenosine Triphosphatases↗
PubMed2005

The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase.

The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.

Adenosine Triphosphatases↗
PubMed2005

NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle-dependent reformation of the ER network.

The endoplasmic reticulum (ER) has a characteristic polygonal structure with hallmark three-way junctions. In a previous paper, we reconstituted the disruption of the pre-existing ER network using mitotic cytosol from HeLa cells in streptolysin O (SLO)-permeabilized CHO-HSP cells (stably expressing GFP-HSP47). In addition, we found that interphase cytosol induced reformation of the disrupted ER network into a continuous network structure. Here, we show that the reformation of the ER network is accomplished through two sequential fusion reactions. The first process is mediated by NSF/alpha and gamma-SNAPs, and involves the generation of typical membranous intermediate structures that connect the disrupted ER tubules. A subsequent fusion is mediated by p97/p47/VCIP135, which has been shown to be required for homotypic fusion events in Golgi cisternae regrowth after mitosis. In addition, we also found that both fusion processes involve the t-SNARE, syntaxin 18.

Adenosine Triphosphatases↗
PubMed2005

[Reevaluation of protective effects of alprostadil on hepatic function in patients undergoing hepatectomy].

BACKGROUND: The protective effect of alprostadil (PGE1), used during hepatectomy, on hepatic function has not been clearly proven. We reevaluated this effect by measuring serum alpha-glutathione S-transferase (aGST), which detects liver injury sensitively. METHOD: Thirty hepatocellular carcinoma patients scheduled for hepatectomy were randomly assigned to control (n=12) and PGE1 (n=10) groups. In the latter group, PGE1 was administered intravenously at a rate of 0.05 microg x kg(-1) x min(-1) during surgery. For measuring alphaGST, arterial blood samples were obtained before anesthesia, following laparotomy, and immediately, 2, 4, and 6 hrs after liver resection. RESULTS: The alphaGST concentrations after liver resection were significantly higher, while mean arterial pressures were significantly lower in the PGE1 group. CONCLUSIONS: Our findings suggest that PGE1 medication during hepatectomy cannot protect hepatic function during and after liver resection.

Aged↗
PubMed2004

Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells.

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.

Animals↗
PubMed2003

Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.

Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong alpha1-antitrypsin is enhanced by overexpression of the ER processing alpha1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded alpha1-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415-422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded alpha1-antitrypsin. We also show that misfolded alpha1-antitrypsin NHK contains labeled Glc1Man9GlcNAc and Man5-9GlcNAc released by endo-beta-N-acetylglucosaminidase H in pulse-chase experiments with [2-3H]mannose. Overexpression of ER ManI greatly increases the formation of Man8GlcNAc, induces the formation of Glc1Man8GlcNAc and increases trimming to Man5-7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man8GlcNAc2 isomer B as previous studies have suggested.

Acetylcysteine↗
PubMed2003

EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin.

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin.

Acetylcysteine↗
PubMed2003

A time-dependent phase shift in the mammalian unfolded protein response.

Unfolded or misfolded proteins in the endoplasmic reticulum (ER) must be refolded or degraded to maintain homeostasis of the ER. The ATF6 and IRE1-XBP1 pathways are important for the refolding process in mammalian cells; activation of these transcriptional programs culminates in induction of ER-localized molecular chaperones and folding enzymes. We show here that degradation of misfolded glycoprotein substrates requires transcriptional induction of EDEM (ER degradation-enhancing alpha-mannosidase-like protein), and that this is mediated specifically by IRE1-XBP1 and not by ATF6. As XBP1 is produced after ATF6 activation, our results reveal a time-dependent transition in the mammalian unfolded protein response: an ATF6-mediated unidirectional phase (refolding only) is followed by an XBP1-mediated bidirectional phase (refolding plus degradation) as the response progresses.

Activating Transcription Factor 6↗
PubMed2002

The Kruppel-like factor Zf9 and proteins in the Sp1 family regulate the expression of HSP47, a collagen-specific molecular chaperone.

In several cells and tissues the synthesis of HSP47, a collagen-specific molecular chaperone in the endoplasmic reticulum, is closely correlated with the synthesis of collagen. We previously reported that the Sp1 binding site at -210 bp in the promoter region and the first and second introns are required for the tissue-specific expression of HSP47 in transgenic mice (Hirata, H., Yamamura, I., Yasuda, K., Kobayashi, A., Tada, N., Suzuki, M., Hirayoshi, K., Hosokawa, N., and Nagata, K. (1999) J. Biol. Chem. 274, 35703-35710). Here, we analyze how these introns influence the transcriptional regulation of the hsp47 gene in BALB/c 3T3 cells, which produce high levels of HSP47. In vitro promoter analysis using a luciferase reporter and gel mobility shift analysis revealed that two cis-acting elements in the first and second introns, BS5-B and EP7-D, respectively, are required for the activation of hsp47 in BALB/c 3T3 cells. Several members of the Kruppel-like factor (KLF) family of proteins were identified as BS5-B-binding proteins by yeast one-hybrid analysis using these elements as baits. One of these proteins, KLF-6/Zf9, binds to the BS5-B element and activates expression of the reporter construct when transfected into cells. Chromatin immunoprecipitation assay analysis revealed that the endogenous KLF-6/Zf9 binds the BS5-B elements that contain the CACCC motif, which is a consensus recognition sequence for other proteins in the KLF family. We also showed that BS5-B and EP7-D are bound by two members of the Sp1 family, Sp2 and Sp3. These results suggest that at least three sequences are required for the constitutive expression of hsp47 in BALB/c 3T3 cells: the -210 bp Sp1 binding site, the BS5-B element in the first intron, and the EP7-D element in the second intron. We suggest that KLF proteins regulate the transcription of hsp47 by binding the BS5-B element in cooperation with Sp2 and/or Sp3.

3T3 Cells↗
PubMed2002

Type XXVI collagen, a new member of the collagen family, is specifically expressed in the testis and ovary.

HSP47 is a collagen-specific molecular chaperone that specifically recognizes and binds to the triple helical domain of various types of collagens. Here we report the cloning of the entire coding region of a novel collagen-like protein by yeast two-hybrid screening of a 17.5-day whole mouse embryo cDNA library using HSP47 as a bait. The cDNA of this protein and its deduced amino acid sequence are 2,690 bp and 438 amino acids long, respectively. The protein contains two clusters of Gly-X-Y collagenous repeats and three noncollagenous domains. Northern blot analysis showed that its mRNA is specifically expressed in the testis and ovary in adult tissues and that expression in these tissues is highest in the neonate. Biochemical characterization of this protein revealed that its proline residues are hydroxylated, it undergoes N-linked glycosylation, it forms trimers, and it is secreted in vitro. Immunohistochemical studies showed that the myoid cells and the pre-theca cells synthesized it in the testis and ovary, respectively, resulting in the accumulation of this protein in the extracellular spaces of these organs. These observations suggest that this protein is a new member of the collagen protein family. We thus designated this protein as type XXVI collagen.

Aging↗