1、二、概論二、概論1、分子細(xì)胞生物學(xué)前沿進(jìn)展與技術(shù)、分子細(xì)胞生物學(xué)前沿進(jìn)展與技術(shù)www.ccs.k12.in.us 生物大分子相互作用生物大分子相互作用Interaction between BiomacromoleculesThe biomacromolecules in cellwww.ccs.k12.in.us ProteinDNARNAOthers The interaction of biomacromolecules is the fundamental basis of any cellular functionProteinothersDNARNAInteractionBy th

2、eir friends, you shall know them!The focuses:u Protein-protein interactionu Protein-DNA interactionu Protein-RNA interactionThe importance of protein-protein interactionProtein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective

3、 of biochemistry, signal transduction and networks.Protein-protein interactions are involved in almost every aspects of biological processes.Protein-protein interactions provide potential drug targets.The methods commonly used for studying protein-protein interaction Chemical crosslinking 化學(xué)交聯(lián)化學(xué)交聯(lián) T

4、agged Fusion Proteins (Pull-down) 沉降實(shí)驗(yàn)沉降實(shí)驗(yàn) Co-immunoprecipitation (Co-IP) 免疫共沉淀免疫共沉淀 Yeast Two-hybrid (Y2H) 酵母雙雜交酵母雙雜交 Fluorescence Resonace Energy Trasfer (FRET) 熒光共振熒光共振能量轉(zhuǎn)移能量轉(zhuǎn)移 Docking 嵌合計(jì)算嵌合計(jì)算 Biacore (Surface Plasmon Resonance, SPR) 表面等離子共表面等離子共振振 Chemical CrosslinkingPrinciple: physically coup

5、ling two proteins with a chemical crosslinkerAdvantage: in vivo Crosslinking reagents:1. formaldehyde2. photo-activated crosslinking reagents L-Photo-Leucine L-Photo-MethionineSubsequent analysis:SDS-PAGE and Western blotting Pull-downGST pull-down, His pull-downWestern blot result of His pull-downE

6、xample: Detecting the interaction between FS and ANG using His pull-down experimentCo-Immunoprecipitation(Western Blot or MS)Example: BAG2 was identified by Co-IP as a CHIP-binding proteinCHIP: carboxyl terminus of Hsp70-interacting protein2005 JBC, 280: 3867338681Yeast Two-hybridPrinciple: the modu

7、lar design of eukaryotic transcription factors, which often have separate DNA-binding and transcriptional activation domains. Transcription factor: DB + AD DB: usually fuse to bait proteinAD: prey protein or a cDNA libray Basis of the Two-Hybrid System.(GIBCO BRL instruction manual) Yeast Two-hybrid

8、Yeast Two-hybrid a gold standard for detecting interactions in vivo advantages:in vivohigh-throughput screenting disadvantages:yeast (cellular environment; protein modification)overexpressed proteinnot working with membrane proteinsneed to be confirmed by other methodsDevelopment of Two-hybrid Techn

9、ique Promegas CheckMate system:works in mammalian cells, more likely to have the proper folding and post-translational modifications split-ubiquitin system: good for membrane proteinsFluorescence Resonance Energy Transfer (FRET)FluorescenceGFP and other FPs (fluorescent proteins)FRET: principle FRET

10、 is a distance-dependent physical process by which energy is transferred nonradiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) The donor emission spectrum must significantly overlap the excitation spectrum of the acceptor 供體發(fā)射波與受體供體發(fā)射波與受體激發(fā)波有重疊激發(fā)波有重疊

11、The donor and acceptor fluorophores must be in favorable mutual orientation 合適的空間取向合適的空間取向 The distance between the donor and acceptor fluorophores must be less than 100 距離距離200,000文獻(xiàn)挖掘高通量XML,ASN.1,BioPAX,CSV,SIF,XML否OntoglyphDIPDIPhttp:/dip.doe-//19,34463,168文獻(xiàn)

12、挖掘MIF,XIN是PPI真實(shí)性評(píng)估IntAcIntAct thttp:/www.ebi.ac.uk/ihttp:/www.ebi.ac.uk/intact/sitentact/site 56,071138,931文獻(xiàn)挖掘PSI-MI是控制詞匯，預(yù)測(cè)pull-down實(shí)驗(yàn)HPRDHPRD// 25,20537,581文獻(xiàn)挖掘PSI-MI否PhosphoMotif FinderMINTMINThttp:/mint.bio.uniromhttp:/mint.bio.uniroma2.it/mint/a2.it/mint/28,

13、186103,808文獻(xiàn)挖掘PSI-MI,Osprey是HomoMINTMIPSMIPShttp:/mips.gsf.de/http:/mips.gsf.de/9001,800文獻(xiàn)挖掘PSI-MI,XML否PEDANTThe usage of PPI resources Protein-protein interactions: binary interactions, molecular complexes The network of interacting proteins Pathways (functional) Evolution of protein-protein intera

14、ctionThe possible interaction network of angiogenin and its partnersu Protein-DNA interactionThe interaction between protein and DNA happens in the nucleustranscription regulation: transcription factorsreplicationrepairepigenetic regulation focus: the interaction between cis-acting factor and trans-

15、acting elementThe methods commonly used for studying protein-DNA interactionNuclease protection assay (DNase I footprinting)In vitro DNA-binding assay (DNA mobility shift assay, or EMSA)Yeast one-hybrid systemChromatin immunoprecipitation (ChIP) assayThe methods commonly used for studying protein-DN

16、A interaction Nuclease protection assay (DNase I footprinting) In vitro DNA-binding assay (DNA mobility shift assay, or EMSA) Yeast one-hybrid systemChromatin immunoprecipitation (ChIP) assayChIP assayApplication: identify regions of the genome with which particular proteins interact.Basic Principle

17、: the binding btw antigen and antibodyAdvantage: in vivo & in native condition; direct protein-DNA binding; genomic-wide screenDisadvantage: requirement for high-quality antibody;noise (artificial interaction by crosslinking)http:/ ChIP-based TechniquesChIP-chip: ChIP combined with high-density

18、oligonucleotide array analysisChIP-Seq:ChIP coupled with next generation DNA sequencing Application: precise, genome-wide mapping of binding sitesNext Generation Sequencing different from the traditional Sanger method (1000 bp) use the power of massive parallel processing to read many smaller length

19、s (25-250 bp) of DNA and then piece them together for a complete picture. Basic procedure of ChIP-Seq experimentNature Reviews Genetics, published online 8 September 2009Basic procedure of ChIP-Seq experimentNature Reviews Genetics, published online 8 September 2009u Protein-RNA interactionu Protein

20、-RNA interactionRNA-Protein Interaction ProtocolsMethods in molecular biology, 2008, 488http:/ and RNA Interaction RNA and protein: two main workhorses in the cell, and function together to accomplish a biological task.usually form RNA-protein complexes or ribonucleoproteins (RNPs). the interaction

21、happens:protect RNA from degradationspliceosomeprocessing of RNA by RNasetranscription regulationtranslationribosomeThe Centrality of RNA linear sequence: a simple source of genetic information secondary structure: shield some sequences while exposing others for recognition, facilitates its interact

22、ions with other molecules. tertiary structure: present surfaces for interactions and contain internal environments that are sufficiently sequestered from solvent that they can promote catalytic reactions. No other macromolecule covers this range of chemical space and functions.The methods for studyi

23、ng RNA-protein interactionRNA based approach: RNA Affinity Chromatography Affinity Oligonucleotide Displacement Strategy Protein based approach:Crosslinking and ImmunoPrecipitationhigh-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP)RNA Affinity ChromatographyMethods in molecular biology. 2008, 488: 1-8 Affinity Oligonucleotide Displ