De novo Selection of Macrocyclic Peptides Binding to TMEPAI family by Means of Random Non-standard Peptides Integrated Discovery System

概要

論

文

概

要 (Thesis Abstract)

○論文題目
(Theme)

De novo Selection of Macrocyclic Peptides Binding to TMEPAI family by Means
of Random Non-standard Peptides Integrated Discovery System
（ラピッドシステムを用いた TMEPAI ファミリー結合環状ペプチドの新規選択）

○指導教員
(Supervisor)

加藤光保 教授
人間総合科学研究科
生命システム医学専攻
(Graduate School of Comprehensive Human Sciences
Doctoral Program in Biomedical Sciences
KATO Mitsuyasu professor)

（所 属） 筑波大学大学院人間総合科学研究科 生命システム医学専攻
(your position) (Graduate School of Comprehensive Human Sciences in University of
Tsukuba, Doctoral Program in Biomedical Sciences)

（氏 名）
(your name)

Li Yongqi

Purpose: TMEPAI family proteins, which is composed of TMEPAI and LDLRAD4, are highly
related to tumorigenic activity in various types of cancer. The study is aimed at discovering
macrocyclic peptides which can bind to both TMEPAI and LDLRAD4 proteins.
Materials and Methods: To construct RaPID (Random non-standard Peptide Integrated
Discovery) system for selecting macrocyclic peptides, several components need to be
prepared on bench. Intracellular domain of TMEPAI and LDLRAD4 proteins constructing with
polyhistidine-tag were produced in E. coli system. DNA library coding random macrocyclic
peptides containing 7-15 repeats of NNK was synthesized by PCR reaction and transcribed to
RNA library with T7 RNA polymerase. The prepared RNA library was then ligated with
puromycin linker on its 3' end. Artificial aminoacyl-tRNA synthetase flexizyme and
tRNAfMetCAU were also produced through transcription of each DNA which was prepared by
PCR reaction. ClAc-D-Tyrosine was charged onto the prepared tRNAfMetCAU with flexizyme
and used to replace Methionine in Flexible in vitro translation (FIT) system. After preparing
all the required components for RaPID system, the selection targeting LDLRAD4 was
performed at first and then the selection targeting TMEPAI followed. Second-generation
sequencing was conducted for identifying selected peptides. Two selected peptides were
chemically synthesized through solid-phase peptide synthesis. Biotin or fluorescein were
conjugated onto the peptides and cyclization was achieved via spontaneous formation of
thioether bond between chloroacetyl group of ClAc-D-Tyrosine and cysteine in the C-terminus
constant sequence of the peptides. After synthesis, purification was conducted by High
Performance Liquid Chromatography. Evaluation of the peptide products was conducted by
Matrix-assisted laser desorption/ionization (MALDI) TOF mass spectrometry. Pull-down of
both TMEPAI and LDLRAD4 proteins with two biotin-labeled peptides was performed. Their
binding kinetics were further assessed by Bio-layer Interferometry. Fluorescein-labeled
peptides were used to image overexpressed TMEPAI and LDLRAD4 in 293T cells. Furthermore,
three-dimensional structure of TMEPAI and LDLRAD4 and their peptide interacting sites were
modeled through bioinformatic platforms.
Results: Proteins of intracellular domain of TMEPAI and LDLRAD4 were produced and RaPID
system was constructed. Two dominant macrocyclic peptides were identified in the two-step
selections and named as LIP (LDLRAD4 interacting peptide)-14 and LIP-13. Fluorescein and
biotin labeled peptides were synthesized and molecular mass of the products were confirmed.
Both LIP-14 and LIP-13 could pull down TMEPAI and LDLRAD4 proteins produced in E. coli and
HEK 293T cells. The binding affinity were measured. KD value of LIP-14 binding to TMEPAI and
LDLRAD4 were 900.9 nM and 961.1 nM, respectively, while KD value of LIP-13 binding to
TMEPAI and LDLRAD4 were 425.4 nM and 130.9 nM. Co-staining TMEPAI family
overexpressed HEK293T cells with LIP-14/13 and fluorescent antibodies showed that both
LIP-14 and LIP-13 have similar staining pattern. In silico interaction modeling suggested that
both LIP-14 and LIP-13 bind to the pocket region of TMEPAI and LDLRAD4 proteins.
Discussion: In this thesis, I described the selection of macrocyclic peptides binding to both
TMEPAI and LDLRAD4 via a two-step selection strategy with RaPID system. The selection
targeting the intracellular domain of LDLRAD4 was performed at first and the selected library
was then applied to target the intracellular domain of TMEPAI. The selected peptides named
as LIP-14 and LIP-13 were tested in vitro and their binding ability were confirmed. The results
indicate that the two-step strategy is applicable for discovering macrocyclic peptides

targeting two protein targets. TMEPAI and LDLRAD4 have similar intracellular domains and
both of them have oncogenic functions in various cell types. Discovering peptides targeting
TMEPAI family may offer new potentials for developing TMEPAI family-related research tools
and new diagnostic and therapeutic tools. In future, modifications will be necessary for
further development. For example, we can consider conjugating a cell-penetration peptide
onto LIP-14/13 to enable them penetrating cell membrane. We can also establish a
Proteolysis Targeting Chimeras (PROTACs) with LIP-14/13 for degrading TMEPAI family
proteins.
Conclusion: Two macrocyclic peptides binding to intracellular domains of both TMEPAI and
LDLRAD4 were selected via RaPID system and confirmed by in vitro assays. Modifications such
as enabling cell penetration will be needed in future to further develop both peptides for
diagnostic and therapeutic applications. ...