Tomoyuki Sumizawa

The Structure of Rat Liver Vault at 3.5 Angstrom Resolution

Vaults are among the largest cytoplasmic ribonucleoprotein particles and are found in numerous eukaryotic species. Roles in multidrug resistance and innate immunity have been suggested, but the cellular function remains unclear. We have determined the x-ray structure of rat liver vault at 3.5 angstrom resolution and show that the cage structure consists of a dimer of half-vaults, with each half-vault comprising 39 identical major vault protein (MVP) chains. Each MVP monomer folds into 12 domains: nine structural repeat domains, a shoulder domain, a cap-helix domain, and a cap-ring domain. Interactions between the 42-turn-long cap-helix domains are key to stabilizing the particle. The shoulder domain is structurally similar to a core domain of stomatin, a lipid-raft component in erythrocytes and epithelial cells.

Apoptosis induced by acrylamide in SH-SY5Y cells

Acrylamide (1–5xa0mM) dose-dependently decreased cell viability in human neuroblastoma cells (SH-SY5Y). The caspase-3 activity and cell population in sub-G1 phase were elevated and peaked on exposure to 3xa0mM acrylamide, while both were less so at higher dose (4 and 5xa0mM). Z-VAD-fmk, a pan-caspase inhibitor, lowered the apparent cytotoxicity of acrylamide. U0126, a specific inhibitor of extracellular signal-regulated protein kinase (ERK) kinase, suppressed the elevation of caspase-3 activities as well as that of sub-G1 population. Thus, although mechanisms other than caspase-dependent apoptosis may be involved, apoptotic process seems to take place in the genesis of toxicity of acrylamide in SH-SY5Y cells through ERK pathway and activation of caspase-3.

Involvement of the extracellular signal-regulated protein kinase pathway in phosphorylation of p53 protein and exerting cytotoxicity in human neuroblastoma cells (SH-SY5Y) exposed to acrylamide

Using human neuroblastoma SH-SY5Y cells, effects of acrylamide on p53 protein and intracellular signal transducting pathways were examined. Acrylamide increased p53, phosphorylated p53, and p53-associated protein murine double minute 2 (MDM2). The phosphorylation of p53 was specific for the Ser15 site. Among mitogen-activated protein kinases (MAPKs), acrylamide caused phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 but not c-Jun NH2-terminal kinase. Nevertheless, blocking p38 pathway by LL-Z1640-2 did not suppress the phosphorylation of p53xa0at Ser15. In contrast, a specific inhibitor of ERK kinase (U0126 or PD98059) could abolish the accumulation as well as the phosphorylation of p53xa0at Ser15. Elevation of MDM2 was also abolished by U0126. An inhibitor of phosphatidylinositol 3-kinase-related kinase (PIKK) pathway (wortmannin) suppressed the increase of p53 and its phosphorylation at Ser15. Hence, acrylamide increases p53 protein and its phosphorylation at Ser15 through ERK and/or PIKK pathways. On the other hand, U0126 and PD98059 suppressed to some extent the cytotoxicity of acrylamide evaluated by trypan blue exclusion and lactate dehydrogenase (LDH) leakage, whereas neither LL-Z1640-2 nor wortmannin was effective in suppressing the toxicity. Thus, ERK pathway seems to play a role both in causing the phosphorylation of p53xa0at Ser15 and in the cytotoxicity of acrylamide in SH-SY5Y cells.

Suppression of acrylamide toxicity by carboxyfullerene in human neuroblastoma cells in vitro

In our previous study, we found that caspase-dependent apoptosis played a role in the genesis of toxicity of acrylamide in human neuroblastoma (SH-SY5Y) cells (Sumizawa and Igisu in Arch Toxicol 81:279–282, 2007). In the present experiment, we examined whether carboxyfullerene may suppress the cytotoxicity of acrylamide because carboxyfullerene has been reported to protect nerve cells from various pathologic processes including apoptosis. Carboxyfullerene lowered lactate dehydrogense leakage and elevated cell viability in SH-SY5Y cells exposed to acrylamide. It also lowered caspase-3 activities and cell population in the sub-G1 phase induced by acrylamide. Nevertheless, carboxyfullerene enhanced cellular uptake of [14C]acrylamide. On the other hand, acrylamide markedly decreased glutathione (GSH)-content in cells and carboxyfullerene blocked the decrease. The toxicity of acrylamide was suppressed by adding GSH or GSH monoethyl ester, whereas it was not lowered by carboxyfullerene when GSH synthesis was inhibited by l-buthionine-(S,R)-sulfoximine. Thus, the cytotoxicity of acrylamide including apoptotic processes is closely related to GSH level in SH-SY5Y cells and carboxyfullerene suppresses the toxicity by maintaining GSH content. Neither tricarboxylic acids without fullerene moiety nor hydroxylated fullerene showed comparable effects of carboxyfullerene (60xa0μM) against 1–5xa0mM acrylamide, suggesting the importance of the three malonic acid groups at specific positions in a fullerene molecule for the effects.

A vault ribonucleoprotein particle exhibiting 39-fold dihedral symmetry

A vault from rat liver was crystallized in space group C2. Rotational symmetry searches indicated that the particle has 39-fold dihedral symmetry.

Hyperosmotic stress up-regulates the expression of major vault protein in SW620 human colon cancer cells

The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP. Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress.

Binding of Human Serum Proteins to Titanium Dioxide Particles In Vitro

Binding of Human Serum Proteins to Titanium Dioxide Particles In Vitro: Mazen S.K. Zaqout, et al. Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan—

A vault ribonucleoprotein particle exhibiting 39‐fold dihedral symmetry. Corrigendum

A corrigendum to the article by Kato et al. [Acta Cryst. (2008). D64, 525–531].

Structure of vault purified from rat liver

Structure of vault purified from rat liver Koji Kato, Hideaki Tanaka, Tomoyuki Sumizawa, Eiki Yamashita, Masato Yoshimura, Yong Zhou, Min Yao, Isao Tanaka, Kenji Iwasaki, Tomitake Tsukihara Osaka University, Institute for Protein Research, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi, Kitakyushu, 807-8555, Japan, Taiwan Beamline Office at SPring-8 1-1-1 Kouto,Sayo-cho, Sayo-gun, Hyogo, Japan, Hokkaido University, Sapporo, 060-0810, Japan, E-mail:k-kato@ protein.osaka-u.ac.jp