通過分析蛋白質(zhì)分子網(wǎng)絡(luò)進(jìn)行靶向分子設(shè)計(jì)

1、通過分析蛋白質(zhì)分子網(wǎng)絡(luò)進(jìn)行靶向分子設(shè)計(jì)計(jì)算與系統(tǒng)生物學(xué)中心September 10, 2010, Wuhan1蔣太交實(shí)驗(yàn)室研究方向病毒序列流感演化的規(guī)律，分子機(jī)理及疫苗與藥物設(shè)計(jì)我實(shí)驗(yàn)室主要通過發(fā)展計(jì)算生物學(xué)與系統(tǒng)生物學(xué)的方法，研究流感病毒流行傳播規(guī)律和分子機(jī)理以及進(jìn)行流感疫苗與藥物設(shè)計(jì)。在計(jì)算方面：發(fā)展的算法主要包括:1、生物分子網(wǎng)絡(luò)的構(gòu)建與模擬2、模擬蛋白質(zhì)結(jié)構(gòu)3、預(yù)測蛋白質(zhì)分子之間的相互作用4、蛋白質(zhì)與多肽藥物分子設(shè)計(jì)在系統(tǒng)實(shí)驗(yàn)方法學(xué)方面：利用與發(fā)展高通量的分子相互作用檢測系統(tǒng)+2基于分子網(wǎng)絡(luò)的靶向分子設(shè)計(jì)BackgroundComplex life phenomenon and sys

2、tems biology molecular network and protein domainsThe coiled-coil motif, an interaction motif of our interestToward mapping of coiled-coil interactome in model organism, yeast using computation and experimentsDesign of coiled-coil switch to regulate cell cycle.3From computer system to biological sys

3、tem 4From computer system to biological system Top-down strategy:DecompositionBottom-up strategy:IntegrationSystems biology5復(fù)雜的生命現(xiàn)象具有復(fù)雜的分子機(jī)制, 形成了一個(gè)復(fù)雜的分子網(wǎng)絡(luò)。（1）腫瘤的發(fā)生P53-centred molecular network（ 2）流感的傳染與復(fù)制Influenza A virus life cycle, a complex process that involves interactions within viral proteins

4、 and between viral proteins and host proteins.6The high throughput technologies drive the development of systems biology.Structural ProteomicsSNP, Comparative GenomicsChip tech = transcriptome Func GenomicsSequencingInteractomeGene/protein network Molecular mechanisms for diseases, metabolism, and o

5、ther physiological activities.Mass spec = complex and modificationComputational modelingOmics What we are interested in :Design principles underlying these complex networks7Scientific problem of our interestCommon Building Blocks: Interaction Motifs/DomainsDomain-level interaction networkstructural

6、basis?How is a protein network organized in terms of structure?8Most proteins are composed of a series of conserved and specialized domainsP539Protein interactions are always mediated by a set of conserved domains or motifsT. Pawson, P. Nash, Science (New York, N.Y 300, 445 (Apr 18, 2003).10Our rese

7、arch goalCommon Building Blocks: Interaction Motifs/DomainsDomain-level interaction network1, understanding of how protein-level network is designed at domain/motif level2, Modulation of protein-level interactions at domain level (= drug design)Domain-based interaction designNetwork modulationStruct

8、ural Basis11The domain/motif of our interest The coiled-coil motif1. Regularity in both sequence and structure2. Prediction of coiled coil by Paircoil21213The coiled-coil motif mediates crucial biological pathwaysKonig & Richmond, JMB 233, 1993 H. M. Strauss, S. Keller, Handb Exp Pharmacol, 461 (200

9、8).SNAREs of membrane fusionbZIP Transcription factor virus entry (HIV: gp41; Flu virus: HA)14Overview of predicted coiled coils in budding yeast1, Coiled coils can be predicted by Paircoil2 with high accuracy2, 10% of yeast proteome contain predicted coiled coils3, Its average size: 63 amino acids4

10、, Coiled coils participate in a variety of cellular processesFunction distributionGenomeCoiled-coil proteins15Mapping coiled-coil interactome of budding yeast using yeast two-hybrid assay.16Coiled-coil networking in known protein complexes17The enrichment of coiled-coil interactions within and betwe

11、en 15 macrocomplexes containing no less than 5 coiled-coil proteins18Coiled-coil-mediated molecular machinesWe define a protein complex as a coiled-coil-mediated molecular machine if it is not only significantly enriched with coiled coils but also significantly enriched with coiled-coil-mediated pro

12、tein interactions.19Kinetochore is essentially a coiled-coil-mediated molecular machinery20Can we design coiled-coil switches to regulate the functions of Kinetochore?First, we look at what happens if we over-express coiled coils in yeast cells.Then, we design an inhibitor coiled-coil to reverse the

13、 effect of the over-expressed coiled coils in yeast. 21 pGAL1-Trp Kinetochre coiled coil :sever as an inhibitor TRP1 GAL1 First, we look at what happens if we over-express coiled coils in yeast cells.Utilizing coiled coils as an inhibitor to disturbing the normal function of kinetochore coiled-coil

14、network.22We found over-expressions of many coiled coils inhibit cell growthcolony formation defect on agar plates Growth curve for Ndc80p-cc1 and Nuf2p-cc2 overexpression +: growth defect upon CC overexpression23Cells were blocked in G2/M phase with 2C DNA content in flow cytometry assay.ControlNdc

15、80p-cc10hr1hr2.5hr5hr7hrOverexpression of kinetochore coiled coil can result in a kinetochore-null phenotype -Cell cycle phase block 24DAPIcc-GFPmergemorphologycontrolNdc80p-cc110% cell with bud36% cell with budCell are elevation of large-budded cells in Morphologic analysis Overexpression of kineto

16、chore coiled coil can result in a kinetochore-null phenotype -Cell budding arrest 25Can we design coiled-coil switches to regulate the functions of Kinetochore?First, we look at what happens if we over-express coiled coils in yeast cells.Then, we design a competitor coiled-coil to reverse the effect

17、 of the over-expressed coiled coils in yeast. 26Design of competitor coiled coil to reverse the processDeveloping an in vivo, artificial, coiled-coil based switch that uses a competitor coiled coil to interact with the inhibitor coiled coil and release cells from inhibitor repressive status .27The c

18、oiled coils that interact with the inhibitor coiled coils can act as competitor coiled coilsNdc80p-cc1 was used as inhibitor coiled coil and his six interaction partners in kinetochore coiled-coil network serving as potential competitor coiled coils. Spc24p-cc2 from Spc24p and Nnf1p-cc1 from Nnf1p c

19、an efficiently rescue the cell from inhibition status. Therefore, the domain-level interactome mapping enables us to design coiled-coil switch to regulate cell functionality.28The coiled-coil switch designed according to yeast coiled-coil interactome could also have effect on human cancer cells (from professor Hang)試驗(yàn)發(fā)現(xiàn)，在只表達(dá)CFP熒光蛋白的細(xì)胞中，能夠找到形成明顯紡錘體的細(xì)胞；在NDC80_CC和NUF2_CC表達(dá)的細(xì)胞中，很難找到明顯有紡錘體形成的細(xì)胞，微管沿質(zhì)膜分布或散亂分布。在穩(wěn)定克隆實(shí)驗(yàn)中，NDC80-CC組無法獲得穩(wěn)定克隆，持續(xù)表達(dá)NDC80-CFP融合蛋白的細(xì)胞不能長期存活?？赡躈DC80片段對細(xì)胞紡錘體的干擾強(qiáng)度大，從而影響了細(xì)胞的生長。變圓突起、帶