Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene-trapped mutant (Arl8b-/- ) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b-/- embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b-/- embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b-/- embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.

How left-right (LR) asymmetric forms in the animal body is a fundamental problem in Developmental Biology. Although the mechanisms for LR asymmetry are well studied in some species, they are still poorly understood in invertebrates. We previously showed that the intrinsic LR asymmetry of cells (designated as cell chirality) drives LR asymmetric development in the Drosophila embryonic hindgut, although the machinery of the cell chirality formation remains elusive. Here, we found that the Drosophila homologue of the Id gene, extra macrochaetae (emc), is required for the normal LR asymmetric morphogenesis of this organ. Id proteins, including Emc, are known to interact with and inhibit E-box-binding proteins (E proteins), such as Drosophila Daughterless (Da). We found that the suppression of da by wild-type emc was essential for cell chirality formation and for normal LR asymmetric development of the embryonic hindgut. Myosin ID (MyoID), which encodes the Drosophila Myosin ID protein, is known to regulate cell chirality. We further showed that Emc-Da regulates cell chirality formation, in which Emc functions upstream of or parallel to MyoID. Abnormal Id-E protein regulation is involved in various human diseases. Our results suggest that defects in cell shape may contribute to the pathogenesis of such diseases.

Helicobacter pylori, a pathogen of various gastric diseases, has many genome sequence variants. Thus, the pathogenesis and infection mechanisms of the H.pylori-driven gastric diseases have not been elucidated. Here, we carried out a large-scale proteome analysis to profile the heterogeneity of the proteome expression of 7 H.pylori strains by using an LC/MS/MS-based proteomics approach combined with a customized database consisting of nonredundant tryptic peptide sequences derived from full genome sequences of 52 H.pylori strains. The nonredundant peptide database enabled us to identify more peptides in the database search of MS/MS data compared with a simply merged protein database. Using this approach, we carried out proteome analysis of genome-unknown strains of H.pylori at as large a scale as genome-known ones. Clustering of the H.pylori strains using proteome profiling slightly differed from the genome profiling and more clearly divided the strains into two groups based on the isolated area. Furthermore, we identified phosphorylated proteins and sites of the H.pylori strains and obtained the phosphorylation motifs located in the N-terminus that are commonly observed in bacteria.

The PUF RNA-binding protein Puf5 is involved in regulation ofthe cell wall integrity (CWI) pathway in yeast. Puf5 negatively regulates expression of LRG1 mRNA, encoding for a GTPase-activating protein for Rho1 small GTPase. Here, we further analyzed the effect of Puf5 on LRG1 expression, together with Ccr4 and Pop2 deadenylases, Dhh1 decapping activator, and other PUF proteins. We found that the growth defect of puf5∆ mutant was enhanced by ccr4∆ mutation, which was partially suppressed by LRG1 deletion. Consistently, Lrg1 protein level was much more up-regulated in ccr4Δ puf5Δ double mutant than in each single mutant. Interestingly, LRG1 poly(A) tail length was longer in ccr4∆ mutant but not in puf5∆ mutant. Thus, Puf5 regulates LRG1 expression independently from Ccr4, although Puf5 recruits the Ccr4-Not deadenylase complex for mRNA destabilization. Unexpectedly, puf6Δ mutation suppressed the growth defect caused by ccr4Δ puf5∆ mutation. Loss of Rpl43a and Rpl43b ribosomal proteins, the previously identified Puf6 interactors, also suppressed the growth defect of ccr4Δ puf5Δ mutant. Our results suggest that Puf5 functions in the CWI pathway by regulating LRG1 expression in a deadenylase-independent manner, and that Puf6 is involved in the Ccr4- and Puf5-mediated regulation of cell growth through association with Rpl43.

To clarify the pathogenic mechanism of disease and establish effective therapies, animal disease models that can be dynamically analyzed are urgently required. Knockout mouse models and conditional genetically engineered mouse models were developed to analyze genes and proteins involved in disease. However, these methods have drawbacks, including embryonic lethality, side effects and low efficiency. To address this issue, we created a novel transgenic mouse model in which the YB1 gene was fused with a destabilizing domain (DD), named the YB1-DD mouse. YB-1 is widely expressed throughout development and has been implicated as a cell survival factor. Newly synthesized DD proteins are degraded through the proteasome pathway, but their degradation can be blocked with trimethoprim (TMP). In this study, we established a novel conditional knockdown mouse model that enables targeting of protein degradation directly; this model resulted in dose-dependent regulation of the target protein YB-1 by the ligand TMP in YB1 heterozygous mice. Since this conditional knockdown mouse model appears to be functional, it has potential as a useful disease model based on direct protein degradation control.

The B-cell receptor (BCR) transmits a tonic survival signal in the absence of antigen stimulation and an antigen-triggered survival signal. Mature B cells express two types of BCR, IgM and IgD, but it remains unclear how B-cell survival is differentially regulated by these two receptors. We found that, whereas cross-linking IgM on spleen B cells greatly enhanced their survival, cross-linking IgD did not enhance, but rather decreased, their survival. Consistently, cross-linking both IgM and IgD only moderately enhanced B-cell survival, suggesting that IgM and IgD play opposing roles in B-cell survival induced by BCR stimulation. Based on these and additional experimental results, we present a mathematical model integrating IgM- and IgD-mediated survival signals. Our model shows that IgD can transmit a tonic survival signal in the absence of antigen stimulation but cross-linking IgD not only does not generate a survival signal but also disrupts its tonic signal, resulting in inhibition of B-cell survival. These results suggest that IgD attenuates BCR-induced survival in mature B cells, presumably to restrain B-cell response to weak and/or self-antigens and prevent nonspecific B-cell activation and autoimmunity.

The proteasome core particle (CP) is a cytoplasmic and nuclear protease complex and is comprised of two α-rings and two β-rings stacked in order of αββα. The assembly of CP proceeds by ordered recruitment of β-subunits on an α-ring with help of assembly chaperones PAC1-PAC2, PAC3-PAC4, and UMP1. However, the mechanism of α-ring formation remains unsolved. Here, we show that α4, α5, α6, and α7 form a core intermediate as the initial process of α-ring assembly, which requires PAC3-PAC4. α1 and α3 can be incorporated independently into the core α4-α7 intermediate, whereas α2 incorporation is dependent on preceding incorporation of α1. Through these processes, PAC1-PAC2 prevents nonproductive dimerization of α-ring assembly intermediates. We also found that PAC1-PAC2 overrides the effect of nuclear localization signals of α-subunits and retains α-ring assembly intermediates in the cytoplasm. Our results first show a detailed assembly pathway of proteasomal α-ring and explain the mechanism by which CP assembly occurs in the cytoplasm.

Histone H2A phosphorylation plays a role both in chromatin condensation during mitosis and in transcriptional activation during the G1/S transition. Bub1 and NHK1/VRK1 have been identified as histone H2A kinases. However, little is known about the importance of histone H2A phosphorylation in chromosome segregation. Here, we expressed recombinant hBUB1 and confirmed that it phosphorylates histone H2A T120 in the in vitro-assembled nucleosome. Knockdown (KD) of BUB1 decreases bulk H2A T120 phosphorylation in HeLa cells, whereas hBUB1 is upregulated during mitosis, which corresponds with H2A T120 phosphorylation. ChIP-qPCR of the DXZ1 centromeric and γ-ALR pericentromeric region showed that BUB1 localizes to this region and increases local H2A T120 phosphorylation during M phase. BUB1 KD did not induce apoptosis but increased the M phase cell population, as detected by flow cytometry. BUB1 KD also caused an abnormal metaphase and telophase, resulting in multinucleated cells and impaired cancer cell growth both in vitro and in vivo. Over-expression of the histone H2A T120D or T120E mutations, which mimic phosphorylated threonine, decreased the number of multinucleated cells caused by BUB1 KD. These results strengthen the apparent importance of BUB1-mediated H2A T120 phosphorylation in normal mitosis.