Publications

Nucleosome compaction facilitates HP1γ binding to methylated H3K9.

The α, β and γ isoforms of mammalian heterochromatin protein 1 (HP1) selectively bind to methylated lysine 9 of histone H3 via their chromodomains. Although the phenotypes of HP1-knockout mice are distinct for each isoform, the molecular mechanisms underlying HP1 isoform-specific function remain elusive. In the present study, we found that in contrast to HP1α, HP1γ could not bind tri-methylated H3 lysine 9 in a reconstituted tetra-nucleosomes when the nucleosomes were in an uncompacted state. The hinge region connecting HP1’s chromodomain and chromoshadow domain contributed to the distinct recognition of the nucleosomes by HP1α and HP1γ.

Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected.

Chemical Synthesis of O-Glycosylated Human Interleukin-2 by the Reverse Polarity Protection Strategy.

 The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.

Manufacturing of chemically defined and xeno-free substrates for cultivation of clinical-grade human iPS cells

Osaka University and Nippi Inc., a Japanese biotech company having an expertise in collagen and collagen-related products, succeeded in manufacturing laminin-511 E8 fragment, a very potent cell-adhesive substrates for cultivation of human pluripotent stem cells, in accordance with GMP (good manufacturing practice). The laminin fragment, commercialized from Nippi under the trade name of “iMatrix-511 MG”, is suitable for production of clinical-grade human iPS cells and other stem cells in cell transplantation therapy. The new product will be commercially available in June 2015. iMatrix-511 GM will accelerate the production of a wide variety of pluripotent stem cells, particularly iPS cells, to be used in regenerative medicine.

Structural basis for amyloidogenic peptide recognition by ​sorLA

SorLA is a neuronal sorting receptor considered to be a major risk factor for Alzheimer’s disease. We have recently reported that it directs lysosomal targeting of nascent neurotoxic ​amyloid-β (​) peptides by directly binding ​. Here, we determined the crystal structure of the human ​sorLA domain responsible for ​ capture, Vps10p, in an unbound state and in complex with two ligands. Vps10p assumes a ten-bladed β-propeller fold with a large tunnel at the center. An internal ligand derived from the ​sorLA propeptide bound inside the tunnel to extend the β-sheet of one of the propeller blades. The structure of the ​sorLA Vps10p-​ complex revealed that the same site is used. Peptides are recognized by ​sorLA Vps10p in redundant modes without strict dependence on a particular amino acid sequence, thus suggesting a broad specificity toward peptides with a propensity for β-sheet formation.

Grease matrix as a versatile carrier of proteins for serial crystallography

Femtosecond X-ray serial crystallography has revolutionized atomic resolution structural investigation by drastically expanding the accessibility to proteins in small-sized crystals, at room temperature and on dynamic reaction. Despite such an enticing opportunity, robust and reliable crystal carrying media are largely lacking. Here, we introduce a grease matrix carrier for protein microcrystals to obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type-3 beyond 2Å resolution under ambient conditions.

Canonical Non-homologous End Joining in Mitosis Induces Genome Instability and Is Suppressed by M-phase-specific Phosphorylation of XRCC4

DNA double-strand breaks (DSBs) can be repaired by one of two major pathways–non-homologous end-joining (NHEJ) and homologous recombination (HR)–depending on whether cells are in G1 or S/G2 phase, respectively. However, the mechanisms of DSB repair during M phase remain largely unclear. In this study, we demonstrate that transient treatment of M-phase cells with the chemotherapeutic topoisomerase inhibitor etoposide induced DSBs that were often associated with anaphase bridge formation and genome instability such as dicentric chromosomes. Although most of the DSBs were carried over into the next G1 phase, some were repaired during M phase. Both NHEJ and HR, in particular NHEJ, promoted anaphase-bridge formation, suggesting that these repair pathways can induce genome instability during M phase. On the other hand, C-terminal-binding protein interacting protein (CtIP) suppressed anaphase bridge formation, implying that CtIP function prevents genome instability during mitosis.

Cold Denaturation of Alpha-Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of β2-microglobulin, Alzheimer’s Aβ1-40/Aβ1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although colddenaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloidstructures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores.