通过分析蛋白质分子网络进行靶向分子设计

1、通过分析蛋白质分子网络进行靶向分子设计计算与系统生物学中心September 10, 2010, Wuhan1蒋太交实验室研究方向病毒序列流感演化的规律，分子机理及疫苗与药物设计我实验室主要通过发展计算生物学与系统生物学的方法，研究流感病毒流行传播规律和分子机理以及进行流感疫苗与药物设计。在计算方面：发展的算法主要包括:1、生物分子网络的构建与模拟2、模拟蛋白质结构3、预测蛋白质分子之间的相互作用4、蛋白质与多肽药物分子设计在系统实验方法学方面：利用与发展高通量的分子相互作用检测系统+2基于分子网络的靶向分子设计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复杂的生命现象具有复杂的分子机制, 形成了一个复杂的分子网络。（1）肿瘤的发生P53-centred molecular network（ 2）流感的传染与复制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)试验发现，在只表达CFP荧光蛋白的细胞中，能够找到形成明显纺锤体的细胞；在NDC80_CC和NUF2_CC表达的细胞中，很难找到明显有纺锤体形成的细胞，微管沿质膜分布或散乱分布。在稳定克隆实验中，NDC80-CC组无法获得稳定克隆，持续表达NDC80-CFP融合蛋白的细胞不能长期存活。可能NDC80片段对细胞纺锤体的干扰强度大，从而影响了细胞的生长。变圆突起、带