Recruitment of tumor necrosis factor receptor-associated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death

Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx ( approximately 1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H(2)O(2) treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H(2)O(2)-induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2-/- and Traf6-/- mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.     

A Rac1/phosphatidylinositol 3-kinase/Akt3 anti-apoptotic pathway, triggered by AlsinLF, the product of the ALS2 gene, antagonizes Cu/Zn-superoxide dismutase (SOD1) mutant-induced motoneuronal cell death

AlsinLF, the product of the ALS2 gene, inhibits Cu/Zn-superoxide dismutase (SOD1) mutant-induced neurotoxicity via its Rho guanine nucleotide-exchanging factor domain. We here identified Rac1, a Rho family small GTPase, as a target for the Rho guanine nucleotide-exchanging factor activity of alsinLF. Rac1 associates with alsinLF. The amount of the GTP form of Rac1 is up-regulated by enforced overexpression of alsinLF. We further found not only that constitutively active Rac1 suppresses motoneuronal cell death induced by SOD1 mutants but also that the neuroprotective activity of alsinLF was completely inhibited by knocking down the endogenous Rac1 expression with small interfering RNA for Rac1, indicating that Rac1 is the major effector for alsinLF-mediated neuroprotection. Such alsinLF/Rac1-mediated neuroprotection occurs specifically against the SOD1 mutant-induced cell death but not against the cell death induced by any other neurotoxic insults in motoneuronal NSC34 cells. We further demonstrated that the alsinLF/Rac1-mediated neuroprotective signal is transmitted to the phosphatidylinositol 3-kinase/Akt anti-apoptotic axis. Among three Akt family proteins, Akt3 is the major downstream mediator for alsinLF/Rac1-mediated neuroprotection, which is specifically effective against SOD1 mutant-induced neurotoxicity.     

Comparative analysis of the cytotoxicity of homopolymeric amino acids

Many human proteins have homopolymeric amino acid (HPAA) tracts, although the physiological significance or cellular effects of their presence is poorly understood. We previously reported that 20 kinds of HPAAs show characteristic intracellular localization and that among those, hydrophobic HPAAs aggregate strongly and form high molecular weight proteins when expressed in cultured cells. In this study, we investigated the cytotoxicity of 20 kinds of HPAAs. HPAA tracts of approximately 30 residues fused to the C-terminus of YFP were expressed in COS-7 cells. Cells expressing homopolymeric-Cys, -Ile, -Leu, and -Val showed low viability in Trypan Blue assay. Caspase-3 activity, which is usually upregulated in dying cells, was determined by measuring the cleavage of the peptide substrate Ac-DEVD-MCA and by detecting the cleaved active form of the caspase-3 by Western blotting. The activity of caspase-3 was drastically elevated in cells expressing those HPAAs which showed low viability in Trypan Blue assay. Interestingly, it was found that there is a correlation between the hydrophobicity of a single amino acid and the cytotoxicity of the corresponding HPAA as a homopolymer. These results indicate that the hydrophobicity of HPAAs may cause cytotoxicity.     

Nucleotide excision repair of 5-formyluracil in vitro is enhanced by the presence of mismatched bases

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. Although this residue is a potentially mutagenic lesion and is removed by several base excision repair enzymes, it is unknown whether fU is the substrate of nucleotide excision repair (NER). Here, we analyzed the binding specificity of XPC-HR23B, which initiates NER, and cell-free NER activity on fU opposite four different bases. The result of the gel mobility shift assay showed that XPC-HR23B binds the fU-containing substrates in the following order: fU:C > fU:T > fU:G > fU:A. Furthermore, in the presence of XPC-HR23B, the dual incision activity was the same as the order of the binding affinity of XPC-HR23B to fU. Therefore, it is concluded that even fU, regarded as a shape mimic of thymine, can be recognized as a substrate of NER incision, and the efficiency depends on instability of the base pair.     

A novel technology allowing immunohistochemical staining of a tissue section with 50 different antibodies in a single experiment.

Immunohistochemical (IHC) examination is frequently necessary for a histological differential diagnosis of tumors. To simplify IHC examination, we have developed a novel device called a "multiplex-immunostain chip (MI chip)." The chip is a panel of antibodies contained in a silicon rubber plate that consists of 50 2-mm-diameter wells. A tissue section slide is placed on the plate and is fastened tightly with a specially designed clamp. The plate with the slide is then turned upside down, which applies the antibodies to the section. This technology allows IHC staining of a tissue section with 50 different antibodies in a single experiment, reducing the time, effort, and expense of IHC analysis. In addition, it enables pathologists to compare expression of multiple antigens on a tissue section simply by changing microscopic fields on a single slide. These features are unique to the MI chip technology. The method requires no expensive instruments. This device can be used in various applications in differential diagnosis of tumors and the field of cell biology.     

The ASK1-MAP kinase pathways in immune and stress responses

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase which plays pivotal roles in stress and immune responses. This review is focused on three major subjects on ASK1: the regulatory mechanisms of the kinase activity, the pathophysiological roles in stress response and innate immunity, and the evolutionary perspectives.     

l-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of l-pantoyl lactone

The 1,2-propanediol (1,2-PD) inducible membrane-bound L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of L-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of L-PL to D-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.