蛋白质分析和蛋白质组学.ppt
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1、蛋白质分析和蛋白质组学proteinRNADNA1protein4 Protein function3 Protein localizationGene ontology(GO):-cellular component-biological process-molecular function1 Molecular biology2 Protein families2视角3和4的介绍:Gene Ontology(GO)Consortium3Gene Ontology 成立的背景成立的背景Year19822005Number of records60244,202,133 GenBankGenB
2、ankEMBLEMBLDDBJDDBJPubMed:PubMed:over 15 million citations over 15 million citations4Whats in a name?nGlucose synthesisnGlucose biosynthesisnGlucose formationnGlucose anabolismnGluconeogenesisnAll refer to the process of making glucose from simpler components5Whats in a name?nThe same name can be us
3、ed to describe different conceptsnA concept can be described using different names Comparison is difficult in particular across species or across databases 6本体(ontology)n计算机科学对自然世界认知的形式化的表示,既是可被计算机表示,解释和利用的知识的形式化的研究即本体。本体是结构化的领域知识,并可以被计算机解释和利用。n实现对生命世界中这些概念理解上的共享,包括从不同的视角,不同的术语分类,不同的主体(人和机器)共享概念-概念化
4、的规范nGene Ontology(GO)协会致力于这样一项工程:编辑一组动态的而又可控的词汇来描述基因和基因产物(主要是蛋白质)不同方面的性质。7Ontologies can be represented as graphs,where the nodes are connected by edges n Nodes=concepts in the ontologyn Edges=relationships between the conceptsnodenodenodeedgeOntology Structure8所有这些蛋白质能做什么?n“功能”太有局限性。n生物学家想知道:每个蛋白质能
5、做什么,属于哪条细胞回路或者为什么细胞需要这个功能,以及在什么地方发生了这样的过程。9Gene Ontology的发起的发起n芽殖酵母基因组数据库(SGD)n果蝇基因组数据库(drosophila genome database,简称FlyBase)n小鼠基因组信息数据库;(mouse genome information database,简称MGDGXD)GO数据库不是以其自身为中心而是依靠外部数据库,这些外部数据库中收录的基因及其产物都将用GO定义的词汇进行注释。因此GO是与时俱进与相互合作的代表,它致力于统一基因及其产物注释的方式。You can visit GO at http:/w
6、ww.geneontology.org.10GO(Gene Ontology)structurenGO isnt just a flat list of biological termsnterms are related within a hierarchy11Hierarchical structuren层次性:is a:上一个概念包括下一个概念,下一个概念是上一个概念的实例。part of:下一个概念是上一个概念的一部分 树松树叶子Part ofIs a12True Path RuleTrue Path Rule:如果下一代的如果下一代的术语可以用于描述此基因产物,术语可以用于描述此基因
7、产物,其上一代术语也可以适用。其上一代术语也可以适用。已糖代谢和单糖合成 己糖合成活性 13DAG有向无环(DAG)Simple hierarchies (Trees)Directed Acyclic GraphsOne or more parentsSingle parent14How does GO work?nWhat does the gene product do?nWhere and when does it act?nWhy does it perform these activities?What information might we want to capture abo
8、ut a gene product?15GO:Three ontologiesWhere does it act?What processes is it involved in?What does it do?Molecular FunctionCellular ComponentBiological Processgene product16Molecular Functionn分子功能描述在分子生物学上的活性,如催化活性或结合活性。nSets of functions make up a biological process.insulin bindinginsulin receptor
9、 activity17Cellular Componentnwhere a gene product acts(细胞中的位置指基因产物位于何种细胞器或基因产物组中(如糙面内质网,核或核糖体,蛋白酶体等)18Biological Processn生物学途径是由分子功能有序地组成的,具有多个步骤的一个过程。(细胞生长和维持、信号传导、嘧啶代谢或配糖基的运输)。cell divisiongluconeogenesisgluconeogenesis19Biological Process20lipocalin21以树状图形式以树状图形式显示的显示的GOGO词汇词汇之间的关系之间的关系 22Perspe
10、ctive 3:Protein localization23proteinPerspective 3:Protein localization24Protein localizationProteins may be localized to intracellular compartments,cytosol,the plasma membrane,or they may be secreted.Many proteins shuttle between multiple compartments.A variety of algorithms predict localization,bu
11、t thisis essentially a cell biological question.很多蛋白质不能被单一地确定存在于细胞一个固定位置上。例如膜联蛋白和小G蛋白家族就转移于胞质和膜之间(有时在胞质内,有时在膜上)。这种转移运动取决于是否有特定的细胞信号存在,例如钙离子。25http:/psort.nibb.ac.jp26http:/www.ch.embnet.org/software/TMPRED.form.html2728Localization of 2,900 yeast proteinsMichael Snyder and colleagues incorporated ep
12、itopetags into thousands of S.cerevisiae cDNAs,and systematically localized proteins(Kumar et al.,2002).See http:/ygac.med.yale.edu for a database including2,900 fluorescence micrographs.29Perspective 4:Protein functionFunction refers to the role of a protein in the cell.We can consider protein func
13、tion from a varietyof perspectives.301.Biochemical function(molecular function)RBP binds retinol,could be a carriern例子:酶 结构蛋白 转运蛋白n细胞中不存在没有任何功能的蛋白。312.Functional assignmentbased on homologyRBPcould bea carriertooOthercarrier proteins增味剂结合增味剂结合蛋白是蛋白是lipocalins的一个成员,的一个成员,也被认为是也被认为是一个载体蛋一个载体蛋白白323.Fun
14、ctionbased on structureRBP forms a calyxX射线晶体衍射显示射线晶体衍射显示RBP形成一个类似茶杯的结构,有一圈形成一个类似茶杯的结构,有一圈疏水氨基酸组成,充当一个配体结合位点疏水氨基酸组成,充当一个配体结合位点334.Function based onligand binding specificityRBP binds vitamin A345.Function based oncellular processDNARNARBP is abundant,soluble,secreted356.Function basedon biological p
15、rocessRBP is essential for vision367.Function based on“proteomics”or high throughput“functional genomics”High throughput analyses show.RBP levels elevated in renal failureRBP levels decreased in liver disease37Functional assignment of enzymes:the EC(Enzyme Commission)systemEC号类别描述酶的数目子类的例子1.-.-.-氧化还
16、原酶10031.1.-.-作用于CH-OH基团1.2.-.-作用于醛类或氧络集团2.-.-.-转移酶10762.1.-.-转移碳基团3.-.-.-水解酶11254.-.-.-裂解酶3565.-.-.-异构酶1566.-.-.-连接酶12638Functional assignment of proteins:Clusters of Orthologous Groups(COGs)39Proteomics:High throughput protein analysisProteomics is the study of the entire collection of proteins enc
17、oded by a genome“Proteomics”refers to all the proteins in a celland/or all the proteins in an organismLarge-scale protein analysis2D protein gelsYeast two-hybridRosetta Stone approach 40Classical biochemical approachIdentify an activityDevelop a bioassayPerform a biochemical purificationStrategies:s
18、ize,charge,hydrophobicityPurify protein to homogeneityClone cDNA,express recombinant proteinGrow crystals,solve structure4142Two-dimensional protein gelsFirst dimension:isoelectric focusing Second dimension:SDS-PAGE434445464748Evaluation of 2D gels(IEF/SDS-PAGE)Advantages:Visualize hundreds to thous
19、ands of proteinsImproved identification of protein spotsDisadvantages:Limited number of samples can be processedMostly abundant proteins visualizedTechnically difficult49Affinity chromatography/mass specBait proteinGST50Affinity chromatography/mass specBait proteinGSTAdd yeast extractProtein complex
20、es bindMost proteins do not bind51Affinity chromatography/mass specBait proteinGSTEluteRun gelMALDI-TOFIdentify complexes52Affinity chromatography/mass specData on complexes deposited in databaseshttp:/http:/www.bind.ca535455The yeast two-hybrid systemReporter geneBait proteinDNA BindingPrey protein
21、DNA activationIsolate and sequence the cDNAof the binding partner you have foundWe will learn about it later when we study protein interaction networks56red=cellular role&subcellular localization of interacting proteins are identical;blue=localiations are identical;green=cellular roles are identical
22、57The Rosetta Stone approachMarcotte et al.(1999)and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes,but occasionally theyare fused into a single gene.By scanning many genomes for examples of“fusedgenes,”several thousand protein-protein pred
23、ictionshave been made.58Yeast topoisomerase IIE.coligyrase BE.coligyrase AThe Rosetta Stone approach59罗赛塔石碑60Gene Fusion(Rosetta stone method)G1:G2:E.coliE.coliYeast Yeast trpA trpBtrpA trpBTryptophan synthase subunits A and B,fused in yeast.Tryptophan synthase subunits A and B,fused in yeast.It is
24、based on the observation that some interacting proteins/domains have homologs in other genomes that are fused into one protein chain,a so-called Rosetta Stone protein.61How many“gene fusions”?3 genomes 3 genomes 88 gene fusions 88 gene fusions179genomes 179genomes?fusions?fusionsMarcotteE.coli:6809Y
25、east:4550262protein1 Molecular biology4 Protein function2 Protein families3 Protein localizationGene ontology(GO):-cellular component-biological process-molecular function63Perspective 2:Protein family,domains and motifs为什么关注蛋白质家族?64基因重复65蛋白质同源序列和家族n在目前所有已知的数据库中均没有发现同源序列的蛋白质。它的其他性质(如跨膜区结构域、磷酸化位点、预测出
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