Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP(C)) into its pathogenic, amyloidogenic isoform (PrP(Sc)) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study, we have examined the effects of LF on PrP(Sc) formation by using cell culture models. Bovine LF inhibited PrP(Sc) accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrP(C) by diminishing its internalization and was capable of interacting with PrP(C) in addition to PrP(Sc). Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities.     

Identification of monomorphic and divergent haplotypes in the 2006-2007 norovirus GII/4 epidemic population by genomewide tracing of evolutionary history

Our norovirus (NoV) surveillance group reported a >4-fold increase in NoV infection in Japan during the winter of 2006-2007 compared to the previous winter. Because the increase was not linked to changes in the surveillance system, we suspected the emergence of new NoV GII/4 epidemic variants. To obtain information on viral changes, we conducted full-length genomic analysis. Stool specimens from 55 acute gastroenteritis patients of various ages were collected at 11 sites in Japan between May 2006 and January 2007. Direct sequencing of long PCR products revealed 37 GII/4 genome sequences. Phylogenetic study of viral genome and partial sequences showed that the two new GII/4 variants in Europe, termed 2006a and 2006b, initially coexisted as minorities in early 2006 in Japan and that 2006b alone had dominated over the resident GII/4 variants during 2006. A combination of phylogenetic and entropy analyses revealed for the first time the unique amino acid substitutions in all eight proteins of the new epidemic strains. These data and computer-assisted structural study of the NoV capsid protein are compatible with a model of antigenic drift with tuning of the structure and functions of multiple proteins for the global outgrowth of new GII/4 variants. The availability of comprehensive information on genome sequences and unique protein changes of the recent global epidemic variants will allow studies of diagnostic assays, molecular epidemiology, molecular biology, and adaptive changes of NoV in nature.     

Rescue of mutant alpha-galactosidase A in the endoplasmic reticulum by 1-deoxygalactonojirimycin leads to trafficking to lysosomes

Active-site-specific chaperone therapy for Fabry disease is a genotype-specific therapy using a competitive inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing alpha-galactosidase A (alpha-Gal A) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in the endoplasmic reticulum (ER). We first established an in vitro translation and translocation system using rabbit reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant alpha-Gal A with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wild-type alpha-Gal A was observed when microsomal vesicles containing wild-type or R301Q alpha-Gal A were isolated and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse fibroblasts showed rapid degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/KO mouse fibroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of alpha-Gal A activity and its protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing R301Q but not in cells expressing the wild-type alpha-Gal A. Observation by immunoelectron microscopy showed that the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the rescue of R301Q from ERAD is a key step for normalization of intracellular trafficking of R301Q.     

Visceral helminths of wild boars (Sus scrofa leucomystax) in Japan, with special reference to a new species of the genus Morgascaridia Inglis, 1958 (Nematoda: Schneidernematidae)

Twenty-nine Japanese wild boars (Sus scrofa leucomystax), collected during the hunting seasons of 2005 and 2006 in the western part of the mainland of Japan (Honshu), were examined for their visceral helminths. Eighteen helminth parasites were prevalent in them, including 17 Nematoda species (Metastrongylus elongatus, Metastrongylus salmi, Metastrongylus asymmetricus, Metastrongylus pudendotechus, Stephanurus dentatus, Gnathostoma doloresi, Physocephalus sexulata, Ascarops strongylina, Capillaria suis, Ascaris suum, Globocephalus samoensis, Globocephalus longimucronatus, Strongyloides ransomi, Trichuris suis, Bourgelatia diducta, Oesophagostomum dentatum, and Oesophagostomum watanabei), and one Cestoda species (Pseudanoplocephala nipponensis). Muscle digestion of the diaphragm (using an artificial gastric juice) of 24 wild boars detected a single diplostomulum of probably Pharyngostomum cordatum. In addition, four female and six male adults of Morgascaridia kugii sp. n. (Nematoda: Schneidernematidae) were recovered from the large intestine of a single wild boar. Examination of an additional 20 samples of the stomach and 27 samples of the large intestine could not find this species. To date, recorded species of the genus Morgascaridia are limited to M. sellsi collected from wild boars in Uganda and Congo several decades ago, and thence, no records of the recovery are available. Morgascaridia kugii sp. n. differed from M. sellsi by smaller body dimensions, shorter distance between the precloacal sucker and the cloaca, smaller sizes of the copulatory spicules and the gubernaculum, and smaller sizes of uterine eggs.     

Crystal structure of tRNA N2,N2-guanosine dimethyltransferase Trm1 from Pyrococcus horikoshii

Trm1 catalyzes a two-step reaction, leading to mono- and dimethylation of guanosine at position 26 in most eukaryotic and archaeal tRNAs. We report the crystal structures of Trm1 from Pyrococcus horikoshii liganded with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine. The protein comprises N-terminal and C-terminal domains with class I methyltransferase and novel folds, respectively. The methyl moiety of S-adenosyl-l-methionine points toward the invariant Phe27 and Phe140 within a narrow pocket, where the target G26 might flip in. Mutagenesis of Phe27 or Phe140 to alanine abolished the enzyme activity, indicating their role in methylating G26. Structural analyses revealed that the movements of Phe140 and the loop preceding Phe27 may be involved in dissociation of the monomethylated tRNA•Trm1 complex prior to the second methylation. Moreover, the catalytic residues Asp138, Pro139, and Phe140 are in a different motif from that in DNA 6-methyladenosine methyltransferases, suggesting a different methyl transfer mechanism in the Trm1 family.     

Structure of a sigma28-regulated nonflagellar virulence protein from Campylobacter jejuni

Campylobacter jejuni, a Gram-negative motile bacterium, is a leading cause of human gastrointestinal infections. Although the mechanism of C.jejuni-mediated enteritis appears to be multifactorial, flagella play complex roles in the virulence of this human pathogen. Cj0977 is a recently identified virulence factor in C. jejuni and is expressed by a σ²⁸ promoter that controls late genes in the flagellar regulon. A Cj0977 mutant strain is fully motile but significantly reduced in the invasion of intestinal epithelial cells in vitro. Here, we report the crystal structure of the major structural domain of Cj0977, which reveals a homodimeric “hot-dog” fold architecture. Of note, the characteristic hot-dog fold has been found in various coenzyme A (CoA) compound binding proteins with numerous oligomeric states. Structural comparison with other known hot-dog fold proteins locates a putative binding site for an acyl-CoA compound in the Cj0977 protein. Structure-based site-directed mutagenesis followed by invasion assays indicates that key residues in the putative binding site are indeed essential for the Cj0977 virulence function, suggesting a possible function of Cj0977 as an acyl-CoA binding regulatory protein.     

Crystal structures of fission yeast histone chaperone Asf1 complexed with the Hip1 B-domain or the Cac2 C terminus

The assembly of core histones onto eukaryotic DNA is modulated by several histone chaperone complexes, including Asf1, CAF-1, and HIRA. Asf1 is a unique histone chaperone that participates in both the replication-dependent and replication-independent pathways. Here we report the crystal structures of the apo-form of fission yeast Asf1/Cia1 (SpAsf1N; residues 1-161) as well as its complexes with the B-domain of the fission yeast HIRA orthologue Hip1 (Hip1B) and the C-terminal region of the Cac2 subunit of CAF-1 (Cac2C). The mode of the fission yeast Asf1N-Hip1B recognition is similar to that of the human Asf1-HIRA recognition, suggesting that Asf1N recognition of Hip1B/HIRA is conserved from yeast to mammals. Interestingly, Hip1B and Cac2C show remarkably similar interaction modes with Asf1. The binding between Asf1N and Hip1B was almost completely abolished by the D37A and L60A/V62A mutations in Asf1N, indicating the critical role of salt bridge and van der Waals contacts in the complex formation. Consistently, both of the aforementioned Asf1 mutations also drastically reduced the binding to Cac2C. These results provide a structural basis for a mutually exclusive Asf1-binding model of CAF-1 and HIRA/Hip1, in which Asf1 and CAF-1 assemble histones H3/H4 (H3.1/H4 in vertebrates) in a replication-dependent pathway, whereas Asf1 and HIRA/Hip1 assemble histones H3/H4 (H3.3/H4 in vertebrates) in a replication-independent pathway.     

Structure of the C-terminal phosphotyrosine interaction domain of Fe65L1 complexed with the cytoplasmic tail of amyloid precursor protein reveals a novel peptide binding mode

Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I beta-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel beta-sheet with the beta5 strand of the PID domain, the binding mode typically observed in the PID/PTB.peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn alpha-helix and interacts extensively with the C-terminal alpha-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB.peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins.     

Reactivity of astrocytes to fibroblast growth factor-1 for biogenesis of apolipoprotein E-high density lipoprotein is down-regulated by long-time secondary culture

We previously showed that astrocytes produce and release fibroblast growth factor-1 (FGF-1) upon 1-month primary and 1-week secondary culture (M/W cells) and stimulate themselves by an autocrine manner to produce apoE-high-density lipoproteins (HDL), closely associated with their generation of apoE-HDL in brain injury. Astrocytes prepared by 1-week primary and 1-month secondary culture (W/M cells), however, expressed FGF-1 as much as M/W cells but produce apoE-HDL much less. The W/M cells conditioned medium in fact contained FGF-1 activity to stimulate astrocytes prepared by 1-week primary and 1-week-secondary culture (W/W cells). FGF-1 did not stimulate W/M cells for apoE-HDL biogenesis while it stimulated W/W cells. Phosphorylation of Akt, ERK and MEK were induced by FGF-1 in W/W cells but not in W/M cells. Finally, fibroblast growth factor receptor-1 in the membrane decreased in W/M cells in comparison to W/W cells. Interestingly, the reactivity of astrocytes to FGF-1 was recovered when W/M cells were transferred to the tertiary culture of 1 week. We concluded that astrocytes decrease their reactivity to FGF-1 for apoE-HDL biogenesis in certain conditions. The findings indicate astrocyte FGF-1 enhances biogenesis of apoE-HDL also by a paracrine mechanism.