Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na-ATPase

Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V₁ portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 μM for ADP or 3.1 μM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA₃-B₃ heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA₃-B₃ forming V₁ (NtpA₃-B₃-D-G) complex independent of nucleotides. The V₁ formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V₁ complex was as high as that of native V₁-ATPase purified from the V₀V₁ complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V₁ complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V₁-ATPase complex.     

Solution structure of the antifreeze-like domain of human sialic acid synthase

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface.     

Solution structure of the SWIRM domain of human histone demethylase LSD1

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.     

AG-dependent 3'-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon

In pre-mRNA splicing, a conserved AG/G at the 3'-splice site is recognized by U2AF(35). A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E(+1)) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF(35) enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF(35) but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10-15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E(+1). Analysis of nine other disease-causing mutations at E(+1) detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3'-splice site that requires U2AF(35) for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3'-splice site that does not require U2AF(35) gives rise to normal splicing. The AG-dependence of the 3'-splice site that we analyzed in disease-causing mutations at E(+1) potentially helps identify yet unrecognized splicing mutations at E(+1).     

SIRPα/CD172a regulates eosinophil homeostasis

Eosinophils are abundant in the lamina propria of the small intestine, but they rarely show degranulation in situ under steady-state conditions. In this study, using two novel mAbs, we found that intestinal eosinophils constitutively expressed a high level of an inhibitory receptor signal regulatory protein α (SIRPα)/CD172a and a low, but significant, level of a tetraspanin CD63, whose upregulation is closely associated with degranulation. Cross-linking SIRPα/CD172a on the surface of wild-type eosinophils significantly inhibited the release of eosinophil peroxidase induced by the calcium ionophore A23187, whereas this cross-linking effect was not observed in eosinophils isolated from mice expressing a mutated SIRPα/CD172a that lacks most of its cytoplasmic domain (SIRPα Cyto(-/-)). The SIRPα Cyto(-/-) eosinophils showed reduced viability, increased CD63 expression, and increased eosinophil peroxidase release with or without A23187 stimulation in vitro. In addition, SIRPα Cyto(-/-) mice showed increased frequencies of Annexin V-binding eosinophils and free MBP(+)CD63(+) extracellular granules, as well as increased tissue remodeling in the small intestine under steady-state conditions. Mice deficient in CD47, which is a ligand for SIRPα/CD172a, recapitulated these phenomena. Moreover, during Th2-biased inflammation, increased eosinophil cell death and degranulation were obvious in a number of tissues, including the small intestine, in the SIRPα Cyto(-/-) mice compared with wild-type mice. Collectively, our results indicated that SIRPα/CD172a regulates eosinophil homeostasis, probably by interacting with CD47, with substantial effects on eosinophil survival. Thus, SIRPα/CD172a is a potential therapeutic target for eosinophil-associated diseases.     

Searching for Genomic Region of High-Fat Diet-Induced Type 2 Diabetes in Mouse Chromosome 2 by Analysis of Congenic Strains

SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (T2dm2sa) on chromosome 2. We then produced the congenic strain (SM.A-T2dm2sa (R0), 20.8–163.0 Mb) and demonstrated that the A/J allele of T2dm2sa impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of T2dm2sa and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for T2dm2sa, we constructed R1 and R2 congenic strains. Both R1 (69.6–163.0 Mb) and R2 (20.8–128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6–128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6–75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (Itga6, Zak, Gpr155, and Mtx2) with non-synonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2.     

Crystal structures of the S6K1 kinase domain in complexes with inhibitors

Ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine protein kinase that plays an important role in the PIK3/mTOR signaling pathway, and is implicated in diseases including diabetes, obesity, and cancer. The crystal structures of the S6K1 kinase domain in complexes with staurosporine and the S6K1-specific inhibitor PF-4708671 have been reported. In the present study, five compounds (F108, F109, F176, F177, and F179) were newly identified by in silico screening of a chemical library and kinase assay. The crystal structures of the five inhibitors in complexes with the S6K1 kinase domain were determined at resolutions between 1.85 and 2.10 Å. All of the inhibitors bound to the ATP binding site, lying along the P-loop, while the activation loop stayed in the inactive form. Compound F179, with a carbonyl group in the middle of the molecule, altered the αC helix conformation by interacting with the invariant Lys123. Compounds F176 and F177 bound slightly distant from the hinge region, and their sulfoamide groups formed polar interactions with the protein. The structural features required for the specific binding of inhibitors are discussed.     

Species richness and species composition of fungal communities associated with cellulose decomposition at different altitudes on the Tibetan Plateau

Aims The aims of this study were to compare the fungal communities developing on cotton strips at three different altitudes on the Tibetan Plateau and to assess the environmental variables influencing them.Methods Cotton strips that had been buried in soil for a year were sampled at three sites at different altitudes (4500, 4950 and 5200 m) located on a southeast-facing slope on the Nyainqentanglha Mountains near Damxung. The fungi on the cotton strips were isolated using a modified washing method. The decomposition abilities and colony growth properties of the major species cultured in pure-culture conditions were investigated and compared. Canonical correspondence analysis (CCA) was used to evaluate the relationships between fungal community composition and environmental variables (altitude, soil depth, soil water content [SWC], plant root mass and gravel content).Important findings A total of 24 species were isolated from the cotton strips, and 12 species occurred frequently and were regarded as major species. The number of fungal species was lower at the 4950-m altitude site than at the other two sites, indicating that not only altitude but also other factors affected the number of species present. All of the major species were able to decompose the cotton strips. In the CCA ordination, automatic forward selection revealed that altitude, SWC and plant root mass significantly affected fungal species composition. Our results suggest that species number and the composition of cellulolytic fungal communities are highly correlated with environmental variables as well as altitude in the alpine meadow on the Tibetan Plateau.