ASK Family Proteins in Stress Response and Disease

Cells are continuously exposed to reactive oxygen species (ROS) generated by aerobic metabolism. Excessively generated ROS causes severe dysfunctions to cells as oxidative stress. On the other hand, there is increasing evidence that ROS plays important roles as a signaling intermediate that induces a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis. To transmit physiological ROS-mediated signals and to adapt to oxidative stress, cells are equipped with various intracellular signal transduction systems, represented by mitogen-activated protein kinase (MAPK) cascades. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream regulator of the stress-activated MAPK cascades and has been shown to play critical roles in ROS-mediated cellular responses. Here, we highlight the roles of members of the ASK family, which consists of ASK1 and newly characterized ASK2, in ROS signaling with their possible involvement in human diseases.     

Responses of Heat Shock Proteins hsp27, αB-Crystallin, and hsp70 in Rat Brain After Kainic Acid-Induced Seizure Activity

We determined the changes in the levels of the mammalian small heat shock protein of 25-28 kDa (hsp27) and the hsp alphaB-crystallin in various regions of rat brain after kainic acid-induced seizure activity by means of specific immunoassays. The levels of hsp27 in the hippocampus and entorhinal cortex were markedly increased and reached a maximum (1.5-2 microg/mg of protein) 2-4 days after the seizure. The levels of hsp27 in these regions were considerably high even 10 days after the seizure. A marked increase in levels of mRNA for hsp27 was also observed in the hippocampus of rats 1-2 days after the seizure. A severalfold increase in the levels of alphaB-crystallin was observed in the hippocampus and entorhinal cortex of rats 2 days after the seizure. However, the maximum levels were <50 ng/mg of protein. The levels of protein sulfhydryl group and glutathione were significantly reduced in the hippocampus of rats at 24 h after the seizure, which might have enhanced the expressions of hsp27 and alphaB-crystallin. The expression of inducible mammalian hsp of 70 kDa (hsp70) was also enhanced in the hippocampus of rats after the seizure, as detected by western and northern blotting analyses. Immunohistochemically, an intensive staining of hsp27 was observed in both glial cells and neurons in the hippocampus, piriform cortex, and entorhinal cortex of rats with kainic acid-induced seizure. However, in the cerebellum, where the receptors for kainic acid are also rich, hsp27 was barely induced in the same rats. This might be due to high levels of the cerebellar calcium-binding proteins parvalbumin and 28-kDa calbindin-D, which might have a protective effect against the kainic acid-inducible damage.     

Fission yeast Stn1 maintains stability of repetitive DNA at subtelomere and ribosomal DNA regions

Telomere binding protein Stn1 forms the CST (Cdc13/CTC1-STN1-TEN1) complex in budding yeast and mammals. Likewise, fission yeast Stn1 and Ten1 form a complex indispensable for telomere protection. We have previously reported that stn1-1, a high-temperature sensitive mutant, rapidly loses telomere DNA at the restrictive temperature due to frequent failure of replication fork progression at telomeres and subtelomeres, both containing repetitive sequences. It is unclear, however, whether Stn1 is required for maintaining other repetitive DNAs such as ribosomal DNA. In this study, we have demonstrated that stn1-1 cells, even when grown at the permissive temperature, exhibited dynamic rearrangements in the telomere-proximal regions of subtelomere and ribosomal DNA repeats. Furthermore, Rad52 and γH2A accumulation was observed at ribosomal DNA repeats in the stn1-1 mutant. The phenotypes exhibited by the stn1-1 allele were largely suppressed in the absence of Reb1, a replication fork barrier-forming protein, suggesting that Stn1 is involved in the maintenance of the arrested replication forks. Collectively, we propose that Stn1 maintains the stability of repetitive DNAs at subtelomeres and rDNA regions.     

Nuclear export of phosphorylated galectin-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs

Galectin-3 (Gal-3), a member of the beta-galactoside binding protein family containing the NWGR antideath motif of the Bcl-2 protein family, is involved in various aspects of cancer progression. Previously, it has been shown that the antiapoptotic activity of Gal-3 is regulated by the phosphorylation at Ser(6) by casein kinase 1 (CK1). Here we questioned how phosphorylation at Ser(6) regulates Gal-3 function. We have generated serine-to-alanine (S6A) and serine-to-glutamic acid (S6E) Gal-3 mutants and transfected them into the BT-549 human breast carcinoma cell line, which does not express Gal-3. BT-549 cell clones expressing wild-type (wt) and mutant Gal-3 were exposed to chemotherapeutic anticancer drugs. In response to the apoptotic insults, phosphorylated wt Gal-3 was exported from the nucleus to the cytoplasm and protected the BT-549 cells from drug-induced apoptosis while nonphosphorylated mutant Gal-3 neither was exported from the nucleus nor protected BT-549 cells from drug-induced apoptosis. Furthermore, leptomycin B, a nuclear export inhibitor, increased the cisplatin-induced apoptosis of Gal-3 expressing BT-549 cells. These results suggest that Ser(6) phosphoryaltion acts as a molecular switch for its cellular translocation from the nucleus to the cytoplasm and, as a result, regulates the antiapoptotic activity of Gal-3.     

TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88

MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.     

Diminished medial prefrontal activity behind autistic social judgments of incongruent information

Individuals with autism spectrum disorders (ASD) tend to make inadequate social judgments, particularly when the nonverbal and verbal emotional expressions of other people are incongruent. Although previous behavioral studies have suggested that ASD individuals have difficulty in using nonverbal cues when presented with incongruent verbal-nonverbal information, the neural mechanisms underlying this symptom of ASD remain unclear. In the present functional magnetic resonance imaging study, we compared brain activity in 15 non-medicated adult males with high-functioning ASD to that of 17 age-, parental-background-, socioeconomic-, and intelligence-quotient-matched typically-developed (TD) male participants. Brain activity was measured while each participant made friend or foe judgments of realistic movies in which professional actors spoke with conflicting nonverbal facial expressions and voice prosody. We found that the ASD group made significantly less judgments primarily based on the nonverbal information than the TD group, and they exhibited significantly less brain activity in the right inferior frontal gyrus, bilateral anterior insula, anterior cingulate cortex/ventral medial prefrontal cortex (ACC/vmPFC), and dorsal medial prefrontal cortex (dmPFC) than the TD group. Among these five regions, the ACC/vmPFC and dmPFC were most involved in nonverbal-information-biased judgments in the TD group. Furthermore, the degree of decrease of the brain activity in these two brain regions predicted the severity of autistic communication deficits. The findings indicate that diminished activity in the ACC/vmPFC and dmPFC underlies the impaired abilities of individuals with ASD to use nonverbal content when making judgments regarding other people based on incongruent social information.     

Effects of 6-keto-prostaglandin E1 on ascitic hepatoma-130 in vivo comparison with chemotherapeutic agents

The inhibitory effect of 6-keto-prostaglandin E1 (pGE1) on the growth and survival of ascitic hepatoma (AH-130) cells in vivo was compared with currently used chemotherapeutic agents. Three days after receiving an intraperitoneal injection of 3 X 10(6) AH-130 tumor cells, Donrhyu rats were injected intravenously or intraperitoneally with one of the following: Thromboxane B2 (TXB2) (0.5 mg/kg), 6-keto-pGE1 (0.5 mg/kg), Mitomycin C (MMC) (1.5 mg/kg), or MMC + 6-keto-pGE1 (1.5 mg/kg + 0.5 mg/kg). The mean survival time, median survival time, and increase of life survival percent (ILS%) during a 60 day period revealed that both 6-keto-pGE1 and 6-keto-pGE1 + MMC significantly inhibited AH-130 tumor cell growth, while TXB2 promoted tumor cell growth. We conclude that 6-keto-pGE1 like anti-tumor agents such as MMC, Diketocoriolin B, Carbazilquinon, Endoxan, and 5-Fluorouracil, can significantly inhibit growth of AH-130 tumor cells in vivo, particularly when administered in combination with the anti-tumor agent MMC.     

Phosphorylated Heat Shock Protein 20 (HSPB6) Regulates Transforming Growth Factor-α-Induced Migration and Invasion of Hepatocellular Carcinoma Cells

Human hepatocellular carcinoma (HCC) is one of the major malignancies in the world. Small heat shock proteins (HSPs) are reported to play an important role in the regulation of a variety of cancer cell functions, and the functions of small HSPs are regulated by post-translational modifications such as phosphorylation. We previously reported that protein levels of a small HSP, HSP20 (HSPB6), decrease in vascular invasion positive HCC compared with those in the negative vascular invasion. Therefore, in the present study, we investigated whether HSP20 is implicated in HCC cell migration and the invasion using human HCC-derived HuH7 cells. The transforming growth factor (TGF)-α-induced migration and invasion were suppressed in the wild-type-HSP20 overexpressed cells in which phosphorylated HSP20 was detected. Phospho-mimic-HSP20 overexpression reduced the migration and invasion compared with unphosphorylated HSP20 overexpression. Dibutyryl cAMP, which enhanced the phosphorylation of wild-type-HSP20, significantly reduced the TGF-α-induced cell migration of wild-type HSP20 overexpressed cells. The TGF-α-induced cell migration was inhibited by SP600125, a c-Jun N-terminal kinases (JNK) inhibitor. In phospho-mimic-HSP20 overexpressed HuH7 cells, TGF-α-stimulated JNK phosphorylation was suppressed compared with the unphosphorylated HSP20 overexpressed cells. Moreover, the level of phospho-HSP20 protein in human HCC tissues was significantly correlated with tumor invasion. Taken together, our findings strongly suggest that phosphorylated HSP20 inhibits TGF-α-induced HCC cell migration and invasion via suppression of the JNK signaling pathway.     

New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase

The consecutive genes BF0771–BF0774 in the genome of Bacteroidesfragilis NCTC 9343 were found to constitute an operon. The functional analysis of BF0772 showed that the gene encoded a novel enzyme, mannosylglucose phosphorylase that catalyzes the reaction, 4-O-β-d-mannopyranosyl-d-glucose + Pi → mannose-1-phosphate + glucose. Here we propose a new mannan catabolic pathway in the anaerobe, which involves 1,4-β-mannanase (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772), finally progressing to glycolysis. This pathway is distributed in microbes such as Bacteroides, Parabacteroides, Flavobacterium, and Cellvibrio.