Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

Beta-amyloid precursor protein (APP) has been reported to be expressed in the CNS from the early stages of development. However, the functional role of APP during early development remains unclear. In the present study, we found that the secreted form of APP (sAPP) significantly enhanced proliferation of neural stem cells. Cells were prepared from 13-day embryonic rat neocortex, which was dissected with a Pasteur pipette to make cell clusters. After 12 h of cultivation in the medium without serum, cells around the centre of the cluster were still nestin-positive proliferative cells, i.e. neural stem cells. To determine whether the proliferation of cells was regulated by sAPP, cultures were treated with recombinant sAPP695, the secreted form of human APP695 produced by yeast. Both DNA synthesis and expression of proliferating cell nuclear antigen markedly increased after 5 h of sAPP695 addition. The enhancement of DNA synthesis by sAPP695 stimulation was blocked by the 22C11 monoclonal antibody specific for the amino-terminal region of sAPP. Then, we examined the effect of the amino-terminal fragment of sAPP and the epitope peptide of 22C11 antibody, and found that both of them also promoted DNA synthesis, suggesting that the amino-terminal region of sAPP is responsible for the biological activity. Our findings indicate the possibility that sAPP enhances proliferation of neural stem cells in vivo and plays an important role during the early CNS development.

We report the first identification of phosphorylation sites of the nucleoprotein (N) of the family Paramyxoviridae. The N protein is known to be the most abundant protein in infected cells; it constructs the N-RNA complex (nucleocapsid) and supports transcription and replication of viral genomic RNA. To determine the role of phosphorylation of the N protein, we expressed the N protein of the HL strain of measles virus (MV) in mammalian cells and purified the nucleocapsid. After separation of the C-terminal region from the core region, phosphorylated amino acids were assayed using MALDI-TOF/TOF and ESI-Q-TOF MS analyses. Two amino acids, S479 and S510, were shown to be phosphorylated by both methods of analysis. Metabolic labeling of the N protein with (32)P demonstrated that these two sites are the major phosphorylated sites within the MV-N protein. In transcriptional analysis using negative-strand minigenomic RNA containing the ORF of the luciferase gene, mutants of each phosphorylation site showed approximately 80% reduction in luciferase activity compared with the wild-type N, suggesting that the phosphorylation of N protein is important in the activation of the transcription of viral mRNA and/or replication of the genome in vivo.

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Genetic studies have implicated amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease. While accumulating lines of evidence indicate that APP has various functions in cells, little is known about the proteins that modulate its biological activity. Toward this end, we employed a two-hybrid system to identify potential interacting factors. We now report that ®bulin-1, which contains repetitive Ca 21 -binding EGF-like elements, binds to APP at its aminoterminal growth factor-like domain, the region that is responsible for its neurotrophic activities. Fibulin-1 expression in the brain is con®ned to neurons, and is not expressed signi®cantly by astrocytes or microglia. Direct binding of ®bulin-1 to the secreted form of APP (sAPP) was demonstrated with a pull-down assay using fragments of both ®bulin-1 fused with glutathione-S transferase and sAPP, produced in bacteria and yeast, respectively. The ®bulin-1/sAPP heteromer was shown to form in the conditioned medium of transfected COS-7 cells. Furthermore, ®bulin-1 blocks sAPP-mediated proliferation of primary cultured rat neural stem cells. These results suggest that ®bulin-1 may play a signi®cant role in modulating the neurotrophic activities of APP. Keywords: Alzheimer's disease, amyloid precursor protein, binding protein, ®bulin-1, neural stem cells, proliferation.Amyloid precursor protein (APP) is an integral membrane glycoprotein expressed in the brain and CNS. Genetic studies implicate APP in the pathogenesis of Alzheimer's disease (AD) via b-amyloid (Ab), which is derived from APP. The nonamyloidogenic metabolism of APP involves an enzymatic cleavage within the Ab sequence, releasing the secreted form of APP (sAPP) (reviewed in Selkoe 1994). A possible role for APP and sAPP in the brain is suggested by a number of observations. In neurons, APP is present at synaptic sites and in vesicular structures within the cell body, axon and dendrites (Koo et al. 1990), and while APP-de®cient mice show decreased locomotor activity and learning impairment (Zheng et al. 1995), overexpression of APP in transgenic mice results in increased numbers of presynaptic terminals (Mucke et al. 1994). In addition, intraventricular administration of sAPP protects hippocampal neurons against ischemic injury (Smith et al. 1994) and enhances memory performance (Roch et al. 1994). Also, in vitro studies show that sAPP enhances neuronal survival, neurite outgrowth, and cell adhesiveness (reviewed in Saitoh and Mook-Jung 1996). Finally, sAPP has been shown to enhance proliferation of neural stem cells (Ohsawa et al. 1999) and thyroid epithelial cells (Pietrzik et al. 1998). In total, these ®ndings suggest that APP, and in particular sAPP, has neurotrophic/growth factor-like functions.Proteins of the APP family share a highly conserved region known as C in the cytoplasmic tail, and two highly conserved regions known as E1 and E2 in the extracellular region (Rosen et al. 1989). Secreted APP includes E1 and E2 regions and has a multidomain structure that may facilita...