GQ0003_17008ohta.indd

Size: px

Start display at page:

Download "GQ0003_17008ohta.indd"

あきひろくまじ
4 years ago
Views:

1 Proteome Letters * * shinya.ohta@kochi-u.ac.jp , Walther Flemming 1977 Uli Laemmli Chromosome Scaffold 1 DNA IIα TopoIIα Structure maintenance of chromosome 2 SMC2 Chromosome Scaffold SMC2 SMC4 Chromosome-associated protein D2 CAP-D2 CAP-H CAP-G 2,3 I SMC2 SMC4 CAP-D3 CAP-H2 CAP-G2 II 4 Kinesin family member 4A Kif4A TopoIIα 5 PMM H3 H3pT3K4me2R8me2 6 H3 Centromere protein A CENP-A 7 CENP-A H CENP-B ,13 Multi-classifier combinational proteomics MCCP nano Random Forest nanorf 2017 Japanese Proteomics Society

Proteome Letters 2 2017 2 60 分裂期染色体をターゲットとしたプロテオミクス解析様々な機能のタンパク質が結合している巨大複合体である染色体の組成の決定を試みる研究は染色体の制御メカニズムを明らかにする上で必須となる近年いくつかのグループにより分裂期染色体プロテオームの決定が試みられたが明確な知見が得られてこなかった 12,13 それは

2 Proteome Letters 分裂期染色体をターゲットとしたプロテオミクス解析様々な機能のタンパク質が結合している巨大複合体である染色体の組成の決定を試みる研究は染色体の制御メカニズムを明らかにする上で必須となる近年いくつかのグループにより分裂期染色体プロテオームの決定が試みられたが明確な知見が得られてこなかった 12,13 それは染色体は非常に大きく複雑な複合体であるため単離が困難であり加えて核膜消失後に大量の細胞質タンパク質が非特異的に結合してしまうことが原因であった我々はニワトリ DT40 細胞より分裂期染色体の単離を試み質量分析でその組成を確認したところ 4,027 種類の染色体タンパク質を同定したこの中には 122 のこれまでに見いだされているほぼ全ての分裂期セントロメア局在タンパク質が含まれており Fig. 1A 4,027 種類の染色体タンパク質に新規の機能的な染色体タンパク質が含まれている可能性が高く存在する一方で他グループの先行研究と同様に非特異的染色体結合タンパク質と考えられるものも含まれていた Fig. 1 Proteomic analysis of mitotic chromosome proteins with Random forest analysis predicting novel proteins of interest (A) The 27 classes of proteins found in chromosomes. (B) The rank of all proteins observed for proteomic abundance, enrichment, retention, SMC2 dependency is plotted as vertical color coded lines (rug plots). Proteins are ranked from the highest (left) to lowest values. Centromere proteins are indicated by longer bars. (C) Separation of the two training sets by RF analysis. The line provides optimal separation of the two training sets. RF analysis of B includes the bioinformatic analysis. (D) Position of novel cloned proteins and remaining uncloned proteins in the same 2d analysis with classification of newly cloned proteins from regions to the left and right of the dividing line, respectively. This figure was modified from Ohta et al., 2010 (15).

3 Proteome Letters Fig. 1A 16 1, ,027 a a 13 a estimated copy number = a*(10 [b*(spectral count)/(protein-specific normalization)] 1) Stable Isotope Labeling with Amino acids in Cell culture SILAC SILAC DT40 1 CENP-A 12 CENP-A 1/15 CCAN Ndc80 Mis Ndc80 Fig. 1B 4 SILAC Enrichment Fig. 1B in vitro Retention SILAC Fig. 1B SMC2 dependency Fig. 1B Ska3 Ska3 dependency 14 Classifier classifier 5 True False Learning set

4 Proteome Letters Decision tree 89 Fig. 1C 34 GFP 30 Fig. 1C 16 2 Fig. 1C MCCP Fuzzy proteomics 15,16 6 DT40 SILAC SMC I II SMC5/ Fig. 2A, B MCCP nanorf 18 Fig. 2A GFP sirna 17 Fig. 2C 7 Hydroxy acid-modified metal oxide chromatography HAMMOC 19 4,274 Fig. 3A, B SILAC 2, CENP-T INCENP Ki-67 TopoIIα Fig. 3C DNA 20 Fig. 3C 8 SILAC k-neighbor

5 Proteome Letters Fig. 2 Nano Random Forest to figure out the novel chromosomal regulation (A) Two-dimensional scatter graph plots of RF scores for (a) CPC versus condensin II and (b) CPC versus condensin I. Key proteins are color coded by category: condensin I, pink; condensin II, purple; CPC, blue; kinetochore, orange; and CCAN, green. (B) Magnification of the part of the heat map depicting the combined results of 5,058 proteins from nanorf targeting condensin, condensin I, condensin II, cohesin, SMC5/6, CPC, histones, CCAN, Nup-Ran complexes, kinetochore, chromosomal scaffolds, and ribosomal proteins. (C) Relative mitotic index of HeLa cells after 28 individual sirna treatments against human orthologues. * in (B) shows the sirna target in. This figure was modified from Ohta et al., 2016 (17). したプロテオミクス解析は染色体構造だけでなく多くの未解明のメカニズムの一端を解き明かす歩みに貢献できると考えている著者に開示すべき利益相反状態は無い文献 1 Adolph KW, Cheng SM, Laemmli UK. Role of nonhistone proteins in metaphase chromosome structure. Cell. 1977;12: Hirano T, Kobayashi R, Hirano M. Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell. 1997;89: Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC. Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev Cell. 2003;5: Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T. Differential contributions of condensin I and condensin II

$(B) Immunoblot analysis of the abundance of chromosomal markers and actin in each fraction. Proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue.$

6 Proteome Letters Fig. 3 Quantitative analysis of mitosis-specific phosphorylation (A) Experimental design for quantification of mitosis-specific phosphorylation. (B) Immunoblot analysis of the abundance of chromosomal markers and actin in each fraction. Proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. Xs: isolated mitotic chromosomes; mitotic WCE: whole-cell extract of mitotic cells; nuclear: nuclear fraction of unsynchronized cells; WCE: whole-cell extract of unsynchronized cells. (C) Heat map showing the population category of the 28 protein categories; clustering tendency is shown at the bottom. This figure was modified from Ohta et al., 2016 (20). to mitotic chromosome architecture in vertebrate cells. Cell. 2003;115: Samejima K, Samejima I, Vagnarelli P, et al. Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα. J Cell Biol. 2012;199: Markaki Y, Christogianni A, Politou AS, Georgatos SD. Phosphorylation of histone H3 at Thr3 is part of a combinatorial pattern that marks and configures mitotic chromatin. J Cell Sci. 2009;122: Tachiwana H, Kagawa W, Shiga T, et al. Crystal structure of the human centromeric nucleosome containing CENP-A. Nature. 2011;476: Hori T, Shang W-H, Toyoda A, et al. Histone H4 Lys 20 monomethylation of the CENP-A nucleosome is essential for kinetochore assembly. Dev Cell. 2014;29: Tanaka Y, Nureki O, Kurumizaka H, et al. Crystal structure of the CENP-B protein-dna complex: the DNA-binding domains of CENP-B induce kinks in the CENP-B box DNA. EMBO J. 2001;20: Nakagawa H, Lee J-K, Hurwitz J, et al. Fission yeast CENP-B homologs nucleate centromeric heterochromatin by promoting heterochromatin-specific histone tail modifications. Genes Dev. 2002;16: Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol. 2007;8: Morrison C, Henzing AJ, Jensen ON, et al. Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate. Nucleic Acids Res. 2002;30: Uchiyama S, Kobayashi S, Takata H, et al. Proteome analysis of human metaphase chromosomes. J Biol Chem. 2005;280: Ohta S, Bukowski-Wills J-C, Wood L, et al. Proteomics of isolated mitotic chromosomes identifies the kinetochore protein Ska3/Rama1. Cold Spring Harb Symp Quant Biol. 2010;75:

7 Proteome Letters Ohta S, Bukowski-Wills J-C, Sanchez-Pulido L, et al. The Protein Composition of Mitotic Chromosomes Determined Using Multiclassifier Combinatorial Proteomics. Cell. 2010;142: Kustatscher G, Hégarat N, Wills KLH, et al. Proteomics of a fuzzy organelle: interphase chromatin. EMBO J. 2014;33: Ohta S, Montaňo-Gutierrez LF, Alves F de L, et al. Proteomics analysis with a nano Random Forest approach reveals novel functional interactions regulated by SMC complexes on mitotic chromosomes. Mol Cell Proteomics. 2016;15: Montano-Gutierrez LF, Ohta S, Kustatscher G, Earnshaw WC, Rappsilber J. Nano Random Forests to mine protein complexes and their relationships in quantitative proteomics data. Mol Biol Cell. 2017;28: Sugiyama N, Masuda T, Shinoda K, Nakamura A, Tomita M, Ishihama Y. Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano- LC-MS/MS in proteomics applications. Mol Cell Proteomics. 2007;6: Ohta S, Kimura M, Takagi S, Toramoto I, Ishihama Y. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins. J Proteome Res. 2016;15:

8 Proteome Letters Proteomics Analysis Understanding the Regulation System of the Mitotic Chromosome Shinya Ohta* * shinya.ohta@kochi-u.ac.jp Department of Biochemistry, Medical School, Kochi University, Kohasu, Oko-cho, Nankoku, Kochi , Japan (Received: October 2, 2017; Revised: October 30, 2017; Accepted: November 8, 2017) Packaging of the DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although intensely studied for over 100 years, mitotic chromosome structure and composition are still yet to be fully elucidated. We developed the multi-classifier combinatorial proteomics (MCCP) and nano Random Forest to reveal the protein composition in mitotic chromosomes purified from chicken DT40 cells. One of the greatest advances from the MCCP approach to analysis of the mitotic chromosome proteome is the ability to combine with genetic knock-out and the stable labeling with amino acids in cell culture. These approaches open the door to the identification of all functional components of mitotic chromosomes despite the adventitious binding of cellular background proteins during mitosis. Furthermore, they can be extended by adding additional classifiers to delineate protein complexes and define functional dependencies between them in the context of intact mitotic chromosomes. This will serve both as a starting point for systematic determination of the full range of functions involved in mitotic chromosome segregation, and as a basis for the development of detailed structural and functional interaction maps of key chromosomal sub-domains. Indeed, we have discovered and characterized several number of novel centromere or chromosomal proteins. Currently we succeeded the identification of 4,274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins with using TiO 2 -based hydroxy acid-modified metal oxide chromatography. In future, phosphoproteome analysis combined with MCCP and nanorf approaches based on machine learning would be useful for the analysis of much more complex hierarchical regulation network on mitotic chromosomes. Keywords: chromatin; mitosis; fuzzy proteomics; machine learning; phosphoproteome; SILAC.

【ＰＤＦ作成用】平成23年度共同研究成果報告書.ren

【ＰＤＦ作成用】平成23年度共同研究成果報告書.ren / HAMMOC 90,000 10 HeLa HAMMOC nano-lc-ms 4,358 90 (MS) HAMMOC hydroxy acid-modi ed metal oxide chromatography 30 9 SILAC (stable isotope labeling by amino acids) HAMMOC 10 HeLa 13 C 15 N Arg 2 H Lys HeLa