Unique proteasome subunit Xrpn10c is a specific receptor for the antiapoptotic ubiquitin-like protein Scythe

The Rpn10 subunit of the 26S proteasome can bind to polyubiquitinoylated and/or ubiquitin-like proteins via ubiquitin-interacting motifs (UIMs). Vertebrate Rpn10 consists of five distinct spliced isoforms, but the specific functions of these variants remain largely unknown. We report here that one of the alternative products of Xenopus Rpn10, named Xrpn10c, functions as a specific receptor for Scythe/BAG-6, which has been reported to regulate Reaper-induced apoptosis. Deletional analyses revealed that Scythe has at least two distinct domains responsible for its binding to Xrpn10c. Conversely, an Xrpn10c has a UIM-independent Scythe-binding site. The forced expression of a Scythe mutant protein lacking Xrpn10c-binding domains in Xenopus embryos induces inappropriate embryonic death, whereas the wild-type Scythe did not show any abnormality. The results indicate that Xrpn10c-binding sites of Scythe act as an essential segment linking the ubiquitin/proteasome machinery to the control of proper embryonic development.     

Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.     

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage

Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance. A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear. To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage.     

Surgical influence of pancreatectomy on the function and count of circulating dendritic cells in patients with pancreatic cancer

Background: Dendritic cells (DCs) are important for an immune surveillance. Myeloid DCs (DC1) are important for an effective antitumor immune system. The function and count of circulating DC1 (cDC1) in hosts with a malignant tumor would be defective. This study focused on analyzing the immunological features of cDC1 in patients with pancreatic cancer during the perioperative period. Materials and methods: Thirty-two pancreatic cancer patients who underwent pancreatectomy and 18 age-matched healthy individuals as controls were enrolled in this study. The perioperative cDC count, the stimulatory capacity of cDC1 against allogeneic T cells and TGF-β1 level in the serum were measured. The cDC count was measured at 12 months after the operation. Results: The preoperative cDC1/cDC2 ratio, cDC1 count, and stimulatory capacity of cDC1 were impaired in patients in comparison to controls (P<0.05). The serum TGF-β1 level was significantly higher in patients than controls (P<0.001). The stimulatory capacity of cDC1 recovered after pancreatectomy (P<0.05). The serum TGF-β1 level significantly decreased after the operation (P<0.05); however, they were still significantly higher than controls (P<0.05). Although the cDC1/cDC2 ratio and the cDC1 count did not increase after the pancreatectomy, they recovered as the controls’ level at 12 months after the pancreatectomy in disease-free patients (P<0.05) and the serum TGF-β1 level in those patients at 12 months after the operation significantly decreased compared with those at the postoperative period (P<0.05). Conclusion: Surgical resection of pancreatic cancer could be associated with improved cDC1 function. When a patient remained disease free, the recovery of cDC1 counts was observed approximately 12 months after pancreatectomy. Further strategy will be needed to improve immune function in patients with pancreatic cancer.     

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.     

Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation

Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.     

Spermosin, a trypsin-like protease from ascidian sperm: cDNA cloning, protein structures and functional analysis.

We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi [Sawada et al. (1984), J. Biol. Chem. 259, 2900-2904; Sawada et al. (1984), Dev. Biol. 105, 246-249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone. The isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides, and an open reading frame encoding 388 amino acids, which corresponds to a molecular mass of 41 896 Da. By sequence alignment, it was suggested that His178, Asp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classified as a novel trypsin family member. The mRNA of preprospermosin is specifically expressed in ascidian gonads but not in other tissues. Purified spermosin consists of 33- and 40-kDa bands as determined by SDS/PAGE under nonreducing conditions. The 40-kDa spermosin consists of a heavy chain (residues 130-388) and a long light chain designated L1 (residues 23-129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 (residues 97-129). The L1 chain contains a proline-rich region, designated L1(DeltaL2) which is lacking in L2. Investigation with the glutathione-S-transferase (GST)-spermosin-light-chain fusion proteins, including GST-L1, GST-L2, and GST-L1(DeltaL2), revealed that the proline-rich region in the L1 chain binds to the vitelline coat of ascidian eggs. Thus, we propose that sperm spermosin is a novel trypsin-like protease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fertilization.     

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocytes accumulating hepatitis B surface antigen

Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.     

BAG-6 is essential for selective elimination of defective proteasomal substrates

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.     

Discovery of novel scaffolds for γ-secretase modulators without an arylimidazole moiety

Gamma-secretase modulators (GSMs) selectively inhibit the production of amyloid-β 42 (Aβ42) and may therefore be useful in the management of Alzheimer’s disease. Most heterocyclic GSMs that are not derived from nonsteroidal anti-inflammatory drugs contain an arylimidazole moiety that potentially inhibits cytochrome P450 (CYP) activity. Here, we discovered imidazopyridine derivatives that represent a new class of scaffold for GSMs, which do not have a strongly basic end group such as arylimidazole. High-throughput screening identified 2-methyl-8-[(2-methylbenzyl)oxy]-3-(pyridin-4-yl)imidazo[1,2-a]pyridine (3a), which inhibited the cellular production of Aβ42 (IC₅₀?=?7.1?µM) without changing total production of Aβ. Structural optimization of this series of compounds identified 5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethylisoindolin-1-one (3m) as a potent inhibitor of Aβ42 (IC₅₀?=?0.39?µM) but not CYP3A4. Further, 3m demonstrated a sustained pharmacokinetic profile in mice and sufficiently penetrated the brain.