Year 2016‎ > ‎Members‎ > ‎

Satoki FUJIWARA

*Abstract*下書き

稀少疾患Rett症候群(RTT)は主に女児に発症する神経発達障害である.主な症状として自閉症やてんかん、アスペルガー症候群を引き起こし,現時点での治療法は対症療法のみである.原因タンパク質の一つにCyclin-dependent kinase-like 5(CDKL5)が同定されており,CDKL5は主に脳の神経細胞の核と細胞質に見られる.現段階では9-302残基の立体構造が解明されており,それ以外の配列の構造予測を行ったところ,およそ960残基までは天然変性領域であり,961-1030残基は構造領域であることが予測された.CDKL5の機能喪失によりシナプス形成や細胞内シグナル伝達機構の異常を引き起こす.人を含む高度な脳を持つ霊長類は,生まれてすぐにシナプスを増大させる特異的な脳の発達過程を持つ.しかし,現時点ではCDKL5の構造や機能とRTTの病態メカニズムの関わりは解明されていない.

 本研究では,アミノ酸残基の保存残基を生物種横断的に予測し,その相同性を見ることで進化に伴う保存残基の推移を導出する進化トレース法を行った.その結果からRTTの症状と関連付けることで進化的な観点から発症のメカニズムを推測した.

KEGG OC ViewerよりヒトのCDKL5のオーソログクラスタを取得し,その中でCDKL5のみを選び,それを進化トレース法に用いるデータセットとした.このデータセットを対象にマルチプルアライメントによる配列整理を行い,UPGMA法による系統樹を作成した.

CDKL5は半索動物から保存されていた.RTTの原因となるミスセンス変異が起こるアミノ酸の約半数は半索動物から保存されており,また,ミスセンス変異するアミノ酸はCDKL5のリン酸化酵素領域に集中している.半索動物から生物は中枢神経を形成するようになる.中枢神経は思考や言語,適応行為といった役割を担っており、CDKL5が原因であるRTTの症状のうち,言語コミュニケーションや知的障害を引き起こす症状に関与すると推察した.

 また,C末端側に霊長類の一部でのみで保存されている配列が確認され,この配列の構造予測を行うと,ほとんどが構造領域であった.霊長類の特異的なシナプス形成の観点とシナプス形成の異常がRTTに関与すること,またRTTが幼少期に発症することから,この配列が霊長類の特異的なシナプス形成に関わりがあると推察した.

*Abstract*下書き*英語ver*

Rare Disease Rett Syndrome (RTT) is a neurodevelopmental disorder mainly occurring in girls. The main symptoms are autism, epilepsy, Asperger's syndrome, and the only treatment currently available is symptomatic treatment. Cyclin-dependent kinase-like 5 (CDKL 5) has been identified as one of the causative proteins, and CDKL 5 is mainly found in the nucleus and cytoplasm of brain neurons. At this stage, the steric structure of 9-302 residues has been elucidated. However, at this time, the relationship between the structure and function of CDKL5 and the pathophysiological mechanism of RTT has not been elucidated.

In this study, evolutionary trace method was conducted to predict the conserved residues of amino acid residues across species, and derive the transition of conserved residues due to evolution by looking at homology. Based on the results, we correlated with the symptoms of RTT and inferred the mechanism of onset from an evolutionary point of view.

We obtained ortholog clusters of human CDKL5 from KEGG OC Viewer, select only CDKL5 among them, and use it as a data set for evolution trace method. Array arrangement by multiple alignment was performed on this data set, and a phylogenetic tree by UPGMA method was created.

CDKL 5 has been preserved from hemi-cord animals. About half of the amino acids in which missense mutations causing RTT occur are preserved from hemi- dogs, and missense mutating amino acids are concentrated in the kinase domain of CDKL5. Living organisms will form central nerves from hemi-cord animals. The central nervous system plays a role such as thinking, language, adaptive act, and it is presumed that among the symptoms of RTT caused by CDKL5, it is involved in symptoms causing language communication and intellectual impairment.

*Abstract 最終版*

Rare Disease Rett Syndrome (RTT) is a neurodevelopmental disorder mainly occurring in girls. The main symptoms are autism, epilepsy, Asperger's syndrome, and the only treatment currently available is symptomatic treatment. Cyclin-dependent kinase-like 5 (CDKL 5), which is found mainly in the nucleus and cytoplasm of brain neurons, has been identified as one of the causative proteins. Dysfunction of CDKL5 causes synapse formation and abnormality of intracellular signaling mechanism. However, at this time, the relationship between the structure and function of CDKL5 and the pathophysiological mechanism of RTT has not been elucidated.

In this research, we use an evolutionary tracing method that predicts evolutionary conserved residues by predicting the conserved residues of amino acid residues across species and seeing their homology, and from an evolutionary perspective RTT The purpose was to infer the mechanism of the onset of the disease.

From the KEGG OC Viewer, we obtained ortholog clusters related to human CDKL5 and selected only CDKL5 among them to prepare a data set to be used for the evolution trace method. Array arrangement by multiple alignment was performed on this data set, and a phylogenetic tree by UPGMA method was created. We also made a structure prediction of CDKL5.

It was confirmed that only 960 residues after amino acid residues are preserved in primates only. As a result of predicting the structure of CDKL5, it was predicted that from 297 residues to 960 residues after the protein kinase is a naturally degenerated region, and it was predicted that the region after the protein kinase was a structural region up to 1030 residues. Furthermore, secondary structure prediction of a primate-specific sequence after 960 residues was performed, and it was predicted that a structure containing two β-sheets and one α-helix was taken. In CDKL5 mutants lacking 325 residues or more of CDKL5, it has been reported that kinase activity against epigenome related protein MeCP2 is enhanced. Therefore, it was suggested that the C-terminal region of CDKL5 suppresses its phosphorylation by the epigenome primate-specific amino acid sequence.