Introduction of Research Center for Allergy and Immunology (RCAI) - - PowerPoint PPT Presentation

Introduction of Research Center for Allergy and Immunology (RCAI)

• What is “Immunogenomics” for?-

Osamu Ohara

RIKEN Research Center for Allergy and Immuology Kazusa DNA Research Institute

Organization of RIKEN Research Center for Allergy and Immunology

研究グループ 研究チーム 研究ユニット

Plasmid preparation robot Liquid dispensing robot Thermal cyclers Capillary DNA sequencers High-resolution fluorescent image scanner DNA microarray system Real-time PCR analyzer Computer data mining system Fluorescent gel image scanner Automated sample preparation robot for MS analysis Mass spec. instruments SPR analysis instrument

Central Instruments for genomics

From Genes to Immune systems

（Gene/Transcript analysis system）

High-throughput DNA sequencers

Expression profiling of tens of thousands of genes Analysis of DNA structure

DNA microarray system

Data integration and Data mining

Genome/protein databases

Availability of comprehensive collections of human/mouse cDNA clones

PC cluster computer RCAI/Kazusa cDNA clones RIKEN FANTOM clones DNA microarray data

From Proteins to Immune Systems

（Protein analysis system）

Nano-LC-MS/MS system MALDI-TOF Mass spectrometer High-throughput sample preparation robot

Separation of proteins Identification of proteins Isolation of proteins of interest

Differential detection of fluorescently labeled proteins 2D gel electrophoresis

Detection of proteins

• f interest

Genome Genome

TR(transcribed region)ome ORFome

DNA microarray SAGE etc. Protein (Ab) microarray 2D gel electrophoresis etc Real-world Transcriptome Real-world Proteome

Cell/Tissue Cell/Tissue

Real-world Biological System Snapshots of transcriptome/proteome

時間軸と空間軸が捨象されている 高い空間・時間依存性をもつ

Immunogenomics Immunogenomics

Focused functional genomics Focused functional genomics for Immunology for Immunology

免疫系に関与する細胞のトランスクリプトーム・プロテオーム 免疫系に関与する細胞のトランスクリプトーム・プロテオーム スナップショットから免疫現象の理解に向かう スナップショットから免疫現象の理解に向かう

ー ーゲノム情報に依拠した免疫システムの理解 ゲノム情報に依拠した免疫システムの理解ー ー

2D gel electrophoretic analysis followed by mass spectrometry

Comprehensive understanding

• f the immune system

Etiology of the immune disorders

Transcriptomic analysis

Proteomic analysis Genome Information

Omics Approaches

Protein Interaction Network

Omicsアプローチとは従来の生命科学研究 のアプローチと何が違うのか？

• データ量？

データの「量」は本質的な違いを与えるものではない。 しかし、しばしば実験手法で便宜的に区別されている 場合が見かけられる。

• 仮説に誘導されているかどうか？

すべての生命現象がゲノムにコードされた遺伝子の振る舞いで記述 されているというドグマの下の仮説形成型のアプローチであるか どうかが、Omicsと従来のアプローチを峻別するものである

情報科学への期待は、正にこうしたOmicsアプローチの 実現へ向けてのものである

癌細胞由来のRNAを鋳型に 逆転写させて調製したCy5 ラベルの１本鎖cDNA 正常細胞由来のRNAを鋳型 に逆転写させて調製した Cy3ラベルの１本鎖cDNA 競合的なハイブリダイゼーション 個別の遺伝子に相当する DNAをアレイ化したもの

DNAマイクロアレイの原理

２つの生理的に異なる状態で発現 レベルの異なっている遺伝子を同定 し、その中の遺伝子に今まで知られ ている機能では予想されない振る舞 いがみられるかどうかを調べる。

包括的解析ではあるが、Omics的な アプローチとはちょっと違うのでは….

The Cycle of Knowledge

IDEAS DATA

Synthesis Induction Abduction Hypothesis Analysis Deduction

Hypothesis-driven vs. Hypothesis-free science

Current needs of real-world immunologists to “Omics” studies

• Fill the gap between the physiological

Fill the gap between the physiological immune responses and the molecular events immune responses and the molecular events

Detection of changes at the mRNA and/or protein levels mRNA → DNA microarray Protein → 2D Gel electrophoresis/MS LC/MS/MS Detection of changes in transcriptional and/or translational states or in chromatin structure

mRNA mRNAと蛋白質レベルは独立な と蛋白質レベルは独立な パラメータである パラメータである

mRNA mRNAレベル 蛋白質の合成速度を規定 レベル 蛋白質の合成速度を規定 蛋白質レベル 合成速度と分解速度のバランスで 蛋白質レベル 合成速度と分解速度のバランスで 決定される 決定される 発現量を包括的に測定しうる 発現量を包括的に測定しうる たんぱく質 たんぱく質レベルの レベルの包括的 包括的な測定は な測定は現在の技術ではまだ 現在の技術ではまだ困難 困難。 。 しかし しかし、もし判れば 、もし判ればそれ それは は生体機能に直結している 生体機能に直結している

両者を追跡することが生命現象の理解には必須 両者を追跡することが生命現象の理解には必須

癌細胞由来のRNAを鋳型に 逆転写させて調製したCy5 ラベルの１本鎖cDNA 正常細胞由来のRNAを鋳型 に逆転写させて調製した Cy3ラベルの１本鎖cDNA 競合的なハイブリダイゼーション 個別の遺伝子に相当する DNAをアレイ化したもの

DNAマイクロアレイの原理

x Y

mRNAレベルの包括的な測定

座標軸（Xi, Yi）のハイブリダイゼー ションシグナルが遺伝子iのmRNA 量に相関する。 今後のバイオ分析機器は、 コンピュータの世界で起こ ったことが再現されていく と言われている。 「ダウンサイジング」が一つの キーワードであり、DNAマイクロ アレイはその先駆けである

Examples of 2D protein map

Protein spots identified by PMF or LC-MS/MS analysis

X (pI) Y (Mw)

Quantities of gene products i, Qi, are expressed by the staining intensities

• f protein spots at the positions (Xi, Yi) on 2D map.

X (pI) Y (Mw)

3479 3478 2490 2582 020918S1WMSS-IEFLS1_20 020918S1KMSS-IEFLS1_20 1.629

• 1.326

2.3 1.734 3.746 1 1 1 1 1 3479

• 1.547
• 1.004
• 2.489
• 2.498
• 3.85

1 1 1 1 1 3478

• 1.249
• 1.745

1.518

• 1.149

1.215 1 1 1 1 1 2582

• 1.163
• 1.745

1.654

• 1.055

1.423 1 1 1 1 1 2490 Volume / Area Height / Area Area Peak Height Volume Volume / Area Height / Area Area Peak Height Volume

• Ref. Spot

② 020918s1wmss-iefls1_20a ① 020918s1kmss-iefls1_20a

Volume

Quantitative protein 2D map of the immune system

Once amounts of proteins are expressed quantitatively, the data can be analyzed in exactly the same way as the DNA microarray data

Differential Fluorescent 2D Protein Map

Cell A Cell B Merge Co-electrophoresed two differentially fluoresecent labeled protein samples

More sensitive than fluorescent protein detection!

Conventional Conventional “ “Omics Omics” ” Routine Routine

• 1. DNA microarray analysis
• 2. 2D gel analysis or LC/MS/MS analysis
• 1. Confirmation of modulated genes by real-time PCR or RNA blotting analysis
• 2. Confirmation of modulated proteins by protein blotting analysis

When changes are observed only at the protein level, they are frequently considered to be biologically significant. However, when the changes at the mRNA level are not accompanied by those at the protein level, their biological significance are ambiguous.

Current concerns in proteomic analysis Current concerns in proteomic analysis

• Low comprehensiveness

DNA microarray ~Tens of thousands of genes less than 1 copy mRNA/cell Protein 2D analysis <1000 protein spots/gel more than 50 fmole/spot

How to obtain protein profiles more comprehensively and more sensitively

Beyond the identification of Beyond the identification of human transcribed sequences human transcribed sequences

Identification and characterization of proteins encoded by the transcripts

Protein primary structure in vivo (post-translational modifications) Cellular and subcellular localization Protein-Protein interaction network Quantitative protein level profiling

What is required to move beyond the What is required to move beyond the identification of transcribed sequences? identification of transcribed sequences?

High-throughput platforms to analyze proteins and their functions

+

A non-redundant collection of cDNAs which can produce authentic proteins

How to fill the gap between the How to fill the gap between the genome and the genome and the proteome proteome

• Preparation of affinity capture reagents

which can specifically detect the gene products of interest with high affinity

Identification and characterization of the gene products in vivo

Complementation of the in vitro results!

Molecular mass and pI Post-translational modifications Interacting protein partners Cellular and subcellular localization Tag High-affinity capture reagent Gene product of interest

Mouse KIAA projects (2002~) Mouse KIAA projects (2002~)

Moving toward “Real-World Proteome” using mouse KIAA cDNAs and antibodies against KIAA proteins Evaluation of RNA editing, alternative forms, and the authenticity of human KIAA cDNAs by comparison of KIAA cDNA sequences from human and mouse and analysis at the protein level. Preparation of affinity reagents specific to KIAA gene products

*This project is supported by JST

Mammalian Genomic data Mammalian Genomic data Analysis of primary structure of proteins

Gel electrophoresis HPLC

Mammalian Mammalian cDNA cDNA data data

Mammalian protein sequence database Mammalian protein sequence database

Protein sequence tags

Conventional Conventional proteome proteome analysis through protein analysis analysis through protein analysis

Kazusa Kazusa DNA Research Institute DNA Research Institute

Mammalian Genomic data Mammalian Genomic data

Gel electrophoresis

Mammalian Mammalian cDNA cDNA data data

Mammalian protein sequence database Mammalian protein sequence database

Proteome Proteome analysis through analysis through cDNA cDNA analysis analysis

Expression Expression-

• ready

ready cDNA cDNA clones clones

Recombinant proteins Recombinant proteins Antibodies against the Antibodies against the recombinant proteins recombinant proteins

Immunodetection Immunodetection Antibody array Antibody array

Kazusa Kazusa DNA Research Institute DNA Research Institute

Pull-down the protein complex

Four members of mammalian protein 4.1 family Four members of mammalian protein 4.1 family

Kazusa Kazusa DNA Research Institute DNA Research Institute

Differential localization of four members of Differential localization of four members of protein 4.1 family in rat cerebellum protein 4.1 family in rat cerebellum

g: Granule cell layer g: Granule cell layer W: white matter W: white matter

Neurofilament Neurofilament H H (neuronal axon staining) (neuronal axon staining) Member of protein 4.1 family Member of protein 4.1 family Kazusa Kazusa DNA Research Institute DNA Research Institute

Analysis of mKIAA protein using antibodies

Western blot Western blot Tissue array Tissue array

Cellular localization Cellular localization

CLSM analysis CLSM analysis

Subcellular Subcellular localizaton localizaton

TEM analysis TEM analysis

Subcellular Subcellular localization localization IP coupled with LC IP coupled with LC-

• MS

MS analysis analysis Interacting proteins Interacting proteins Post Post-

• translational modifications

translational modifications

Specific Antibodies

Protein size Alternative form Tissue abundance

Omicsアプローチの実現のために 求められているもの

1. 共通基盤に立った、相互比較可能な包括的解析 データリソースの確立 2. データマイニングのための情報科学的なプラット フォーム

データフォーマットの標準化、コントロールされた用語の使用など Focused genomicsで求められているのは、目的指向型の「仮説形成 推理」を成立させるためのプラットフォーム 解析手法の革新：まだまだ包括的解析の技術レベルは改良されなければ ならない！

Theory of Enlightened Self Theory of Enlightened Self-

• Interest

Interest

“A genome enthusiast is a genome critic who just got a hit in the EST database”

From “Lessons Learned, Promises Kept: A Biologist’s Eye View of the Genome Project”

S.M. S.M.Tilghman Tilghman Genome Res. 1996 6, 773 Genome Res. 1996 6, 773-

• 80

80

Theory of Enlightened Self Theory of Enlightened Self-

• Interest

Interest

A modified version for Immunoinformatics

“An informatics enthusiast is an informatics critic who just got an immunological insight from hypothesis-free science”

Thanks to my colleagues! Thanks to my colleagues!

• Immunogenomics Team (RCAI)

Kitamura, H., Kimura, Y., Nishigaki, T., Murahashi, Y., Nakagawa, T., Yokoyama, R., Ishizu, Y., Takayama, M., Nomura, Y.

• Kazusa DNA Research Institute

Nagase, T., Kikuno, R., Nakayama, M., Yamakawa, H. and many excellent technicians

• JST CREATE Program

Koga, H., Okazaki, N., Ohara, R., Yuasa, S., and many collaborators and excellent technicians