Takashi Umehara

Name:

Organization: Riken , Japan

Department:

Title: (PhD)

Chemicals
References

Development and crystallographic evaluation of histone H3 peptide with N-terminal serine substitution as a potent inhibitor of lysine-specific demethylase 1

Co-reporter:Yuichi Amano, Masaki Kikuchi, Shin Sato, Shigeyuki Yokoyama, Takashi Umehara, Naoki Umezawa, Tsunehiko Higuchi

Bioorganic & Medicinal Chemistry 2017 Volume 25, Issue 9(Issue 9) pp:

Publication Date(Web):1 May 2017

DOI:10.1016/j.bmc.2017.03.016

Lysine-specific demethylase 1 (LSD1/KDM1A) is a flavoenzyme demethylase, which removes mono- and dimethyl groups from histone H3 Lys4 (H3K4) or Lys9 (H3K9) in complexes with several nuclear proteins. Since LSD1 is implicated in the tumorigenesis and progression of various cancers, LSD1-specific inhibitors are considered as potential anti-cancer agents. A modified H3 peptide with substitution of Lys4 to Met [H3K4M] is already known to be a potent competitive inhibitor of LSD1. In this study, we synthesized a series of H3K4M peptide derivatives and evaluated their LSD1-inhibitory activities in vitro. We found that substitutions of the N-terminal amino acid with amino acids having a larger side chain were generally not tolerated, but substitution of Ala1 to Ser unexpectedly resulted in more potent inhibitory activity toward LSD1. X-ray crystallographic analysis of H3K4M derivatives bound to the LSD1·CoREST complex revealed the presence of additional hydrogen bonding between the N-terminal Ser residue of the H3 peptide derivative and LSD1. The present structural and biochemical findings will be helpful for obtaining more potent peptidic inhibitors of LSD1.Download high-res image (262KB)Download full-size image

Multiple Site-Specific Installations of N-Monomethyl-L-Lysine into Histone Proteins by Cell-Based and Cell-Free Protein Synthesis

Co-reporter:Dr. Tatsuo Yanagisawa;Mihoko Takahashi;Dr. Takahito Mukai;Shin Sato;Masatoshi Wakamori;Dr. Mikako Shirouzu;Dr. Kensaku Sakamoto;Dr. Takashi Umehara; Dr. Shigeyuki Yokoyama

ChemBioChem 2014 Volume 15( Issue 12) pp:

Publication Date(Web):

DOI:10.1002/cbic.201402291

Abstract

Lysine methylation is one of the important post-translational modifications of histones, and produces an N^ε-mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N^ε-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N^ε-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N^ε-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA^Pyl) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N^ε-monomethyllysine residues. We installed N^ε-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N^ε-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N^ε-monomethyllysine residue at position 27 better than the N^ε-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N^ε-monomethyllysines at multiple positions within a protein for large-scale production.

Activation of lysine-specific demethylase 1 inhibitor peptide by redox-controlled cleavage of a traceless linker

Co-reporter:Yuichi Amano, Naoki Umezawa, Shin Sato, Hisami Watanabe, Takashi Umehara, Tsunehiko Higuchi

Bioorganic & Medicinal Chemistry (1 February 2017) Volume 25(Issue 3) pp:

Publication Date(Web):1 February 2017

DOI:10.1016/j.bmc.2016.12.033

We have previously employed cyclization of a linear peptide as a strategy to modulate peptide function and properties, but cleavage to regenerate the linear peptide left parts of the linker structure on the peptide, interfering with its activity. Here, we focused on cyclization of a linear peptide via a “traceless” disulfide-based linkage that would be cleaved and completely removed in a reducing environment, regenerating the original linear peptide without any linker-related structure. Thus, the linker would serve as a redox switch that would be activated in the intracellular environment. We applied this strategy to a lysine-specific demethylase 1 (LSD1) inhibitor peptide 1. The resulting cyclic peptide 2 exhibited approximately 20 times weaker LSD1-inhibitory activity than peptide 1. Upon addition of reducing reagent, the linker was completely removed to regenerate the linear peptide 1, with full restoration of the LSD1-inhibitory activity. In addition, the cyclic peptide was far less susceptible to proteolysis than the linear counterpart. Thus, this switch design not only enables control of functional activity, but also improves stability. This approach should be applicable to a wide range of peptides, and may be useful in the development of peptide pharmaceuticals.