Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p

RNase L and Ire1p are members of a superfamily of regulated endoribonucleases that play essential roles in mediating diverse types of cellular stress responses. 2'-5' oligoadenylates, produced in response to interferon treatment and viral double-stranded RNA, are necessary to activate RNase L. In contrast, unfolded proteins in the endoplasmic reticulum activate Ire1p, a transmembrane serine/threonine kinase and endoribonuclease. To probe their similarities and differences, molecular properties of wild-type and mutant forms of human RNase L and yeast Ire1p were compared. Surprisingly, RNase L and Ire1p showed mutually exclusive RNA substrate specificity and partially overlapping but not identical requirements for phylogenetically conserved amino acid residues in their nuclease domains. A functional model for RNase L was generated based on the comparative analysis with Ire1p that assigns novel roles for ankyrin repeats and kinase-like domains.     

Chemo-enzymatic synthesis of 3-(2-naphthyl)- L-alanine by an aminotransferase from the extreme thermophile, Thermococcus profundus

Hyper-thermostable aminotransferase from Thermococcus profundus (MsAT) was used to synthesize 3-(2-naphthyl)-l-alanine (Nal) by transamination between its corresponding -keto acid, 3-(2-naphthyl)pyruvate (NPA) and l-glutamate (Glu) at 70 °C. Equilibrium of this reaction was shifted toward Nal production due to its low solubility, giving rise to Nal precipitate. Optically pure Nal (>99% ee) was synthesized with 93% (mol mol^–1) yield from 180 mM NPA and 360 mM Glu.     

Genomic rearrangements resulting in PLP1 deletion occur by nonhomologous end joining and cause different dysmyelinating phenotypes in males and females

In the majority of patients with Pelizaeus-Merzbacher disease, duplication of the proteolipid protein gene PLP1 is responsible, whereas deletion of PLP1 is infrequent. Genomic mechanisms for these submicroscopic chromosomal rearrangements remain unknown. We identified three families with PLP1 deletions (including one family described elsewhere) that arose by three distinct processes. In one family, PLP1 deletion resulted from a maternal balanced submicroscopic insertional translocation of the entire PLP1 gene to the telomere of chromosome 19. PLP1 on the 19qtel is probably inactive by virtue of a position effect, because a healthy male sibling carries the same der(19) chromosome along with a normal X chromosome. Genomic mapping of the deleted segments revealed that the deletions are smaller than most of the PLP1 duplications and involve only two other genes. We hypothesize that the deletion is infrequent, because only the smaller deletions can avoid causing either infertility or lethality. Analyses of the DNA sequence flanking the deletion breakpoints revealed Alu-Alu recombination in the family with translocation. In the other two families, no homologous sequence flanking the breakpoints was found, but the distal breakpoints were embedded in novel low-copy repeats, suggesting the potential involvement of genome architecture in stimulating these rearrangements. In one family, junction sequences revealed a complex recombination event. Our data suggest that PLP1 deletions are likely caused by nonhomologous end joining.     

Phosphorylated alpha-synuclein is ubiquitinated in alpha-synucleinopathy lesions

alpha-Synuclein is one of the major components of intracellular fibrillary aggregates in the brains of a subset of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz disease, which are referred to as alpha-synucleinopathies. We have shown previously (Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg, M. S., Shen, J., Takio, K., and Iwatsubo, T. (2002) Nat. Cell Biol. 4, 160-164) that alpha-synuclein deposited in synucleinopathy brains is extensively phosphorylated at Ser-129 and migrates at 15 kDa. Here we examined the biochemical characteristics of the additional, higher molecular mass species of phosphorylated alpha-synuclein-positive polypeptides that also are recovered in the Sarkosyl-insoluble fraction of synucleinopathy and migrate at about 22 and 29 kDa. These 22 and 29 kDa bands were positive for three different anti-ubiquitin antibodies and comigrated perfectly with in vitro ubiquitinated alpha-synuclein that may correspond to mono- and diubiquitinated alpha-synuclein, respectively. Furthermore, cyanogen bromide cleavage of the 22 and 29 kDa polypeptides shifted the mobility to 19 and 26 kDa, respectively, and they retained immunoreactivity for both ubiquitin and alpha-synuclein. Finally, protein sequence analysis showed that the 19 kDa band contained two amino-terminal sequences of alpha-synuclein and ubiquitin. These results strongly suggest that phosphorylated alpha-synuclein is targeted to mono- and diubiquitination in synucleinopathy brains, which may have implications for mechanisms of these diseases.     

Radiation-induced chain reactions in alcohol solutions of diphenyliodonium salts: a high-sensitivity chemical dosimeter

Chain reactions in gamma-irradiated 2-propanol solutions of diphenyliodonium salts have been studied. Protonic acids were generated in the irradiated solutions with high yields, whereas acid formation as a result of thermal reactions was negligible. The solution can be used as a high-sensitivity chemical dosimeter. The G value of acidic protons increases with increasing concentration of diphenyliodonium salt at the lower concentrations because the reaction rate of a propagation reaction increases. However, the chain is limited by a termination reaction between phenyl radical and the iodonium salt: The G value shows a maximum value of 610 micromol J(-1) at the concentration of 0.08 mol dm(-3) and decreases at higher salt concentrations.     

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.