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Development of a novel method to analyze 5-hydroxymethylcytosine in the CpG sequence of the genome using maintenance DNA methyltransferase, DNMT1.

In mammals, cytosine in the CpG sequence is often methylated by DNA methyltransferase (Dnmt). Methylated cytosine (5mC) plays crucial roles in gene silencing, genomic imprinting, X-chromosome inactivation and the stability of genomic DNA. Aberrant DNA methylation causes embryonic lethality and cancer. Recently, 5-hydoxymethylcytosine (5hmC) was discovered to be a new modification of cytosine, being an intermediate of the demethylation process, and thus is implicated in the pluripotency of stem cells, development, and disease. Therefore, it is quite important to develop the technique to analyze the position of 5hmC in genome. To determine 5hmC at single-base resolution, several techniques have been reported. However, each method has advantages and disadvantages.

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.