博士后研究工作报告.docx
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博士后研究工作报告
封面典范
分类号___________密级____________
UDC_____________编号____________
中国科学院生物物理研究所
博士后研究工作报告
肿瘤克制有关蛋白RBBP1各构造域的构造与功能研究
宫维斌
工作达成日期
2009
年10
月——2011年11
月
报告提交日期
2011
年11月
中国科学院生物物理研究所（北京）
2011年11月
题名页典范
肿瘤克制有关蛋白RBBP1各构造域的构造与功能研究
Structuralandfunctionalstudiesofthedomainsofatumor
suppressionrelativeprotein,RBBP1
博士后姓名宫维斌
流动站（一级学科）名称生物学
专业（二级学科）名称生物物理
研究工作开端时间
2009
年10
月28
日
研究工作期满时间
2011
年11
月10
日
中国科学院生物物理研究所
2011年11月
中文纲要典范
内容纲要
RBBP1蛋白与RB家族蛋白特异性互相作用，克制细胞增殖有关基因的转
录和表达。
别的，RBBP1与其同源蛋白RBBP1L1也参加表观遗传学调控，并克制乳腺癌、白血病等疾病。
所以，对RBBP1的研究拥有重要的生物学意义和潜伏的医药应用远景。
本文对RBBP1进行了一系列的构造和功能的研究。
RBBP1蛋白包括TD、PD（构造已获得分析）、AD、CD和R2D等5个结
构域，。
本文经过分子生物学方法和核磁共振方法发现：
R2D构造域不行溶；
AD构造域可溶不稳固；TD、PD和CD构造域能够获得较高浓度的蛋白样品，
而且它们皆能够独立折叠，相互之间没有互相作用。
TD，PD和CD构造域都属于“royalfamily”家族，该家族主要联合赖氨
酸和精氨酸甲基化的组蛋白。
本文发现，RBBP1TD和PD构造域不联合
Lys-me1,Lys-me2,Lys-me3以及Arg-me2也不辨别H3K9me3,H3K27me3,
H3K36me3和H4K20me3等甲基化组蛋白。
而CD构造域联合上述各底物，并
且对H4K20me3拥有最强亲和力。
所以，CD构造域是RBBP1辨别甲基化组蛋白的重点构造域。
我们进一步分析了CD构造域的构造，并经过点突变研究确认了其活性位点残基。
CD构造域拥有典型的chromobarrel构造，并形成了该构造域中守旧的芳环盒子，可辨别甲基化组蛋白。
我们也分析了TD构造域的构造，发现这是一个少见的交错双Tudor构造，与组蛋白去甲基化酶JMJD2A的双Tudor构造近似，而它们的序列同源性低，且拥有不一样的功能。
重点词：
肿瘤克制有关蛋白，蛋白质构造，核磁共振，蛋白质-蛋白质互相作用，甲基化组蛋白
英文纲要典范
Abstract
RBBP1specificallyinteractswithRBproteinfamily,suppressingtranscriptionandexpressionofcell-growingrelatedgenes.Moreover,RBBP1anditshomologyproteinRBBP1L1alsoattendepigeneticregulationandfunctionasleukemiaandtumorsuppressor.Sincetheyarebiologicallyimportant,weperformedaseriesofstructuralandfunctionalstudieswithRBBP1,whichwillhelpfuturemedicalstudies.
RBBP1proteinhas5domains,includingTD,PD（structureresolved）,AD,CDandR2D.Inthisstudy,wefoundthatR2DdomainisinsolubleandADdomainissoluble
butnotstable.TheTD,PDandCDdomainsaresolubleandstableathighconcentration.Thesethreedomainscanfoldindependentlywithoutanyinteractionwitheachother.
TD,PDandCDdomainsallbelongtotheRoyalFamily,whichmaybindmethylatedhistonetailsonLysandArgresidues.WefoundthatbothTDandPDdomainslackabilitytobindmethylatedLys-me1,Lys-me2,M3L,Arg-me2,andtri-methylatedhistonetails（H3K9me3,H3K27me3,H3K36me3,andH4K20me3）,whileCDdomainbindsallsubstratesabove,withstrongestbindingaffinityforH4K20me3.TheseresultsdemonstratethatCDdomainofRBBP1isresponsibleforrecognizingmethylatedhistonetailsinchromatinremodelingandepigeneticregulation.WefurthersolvedthesolutionstructureofCDdomain,andconfirmitsactivesitebymutagenesisstudy.CDdomainhasatypicalchromobarrelstructure,andhasthearomaticcageconservedinthechromobarreldomains,whichcanrecognizethemethylatedhistones.
WealsodeterminedthesolutionstructureofRBBP1TDdomain,whichsupprisinglyrepresentsaninterdigitateddoubleTudordomainsimilartoJMJD2A,aJmjcdomain–containinghistonedemethylase,althoughtheyonlyshareverylowsequencehomologyandhavedifferentfunctions.
英文纲要典范
Keywords:
RBBP1,proteinstructure,nuclearmagneticresonance,protein-protein
interaction,methylatedhistone
英文纲要典范
目录典范
第1章绪论······································1
1.1RBBP1的生物学功能·································1
1.2RBBP1的序列剖析与构造域构成························3
1.3Royalfamily····································10
组蛋白修饰与组蛋白密码····························11
研究的主要内容和意义·······························13
第2
章RBBP1各构造域的表达纯化和功能研究··················15
资料与方法····································15
2.1.1RBBP1各构造域的克隆
·························15
2.1.2RBBP1TD、PD和CD
构造域的表达与纯化···············16
2.1.2核磁样品··································18
2.1.3RBBP1TD，PD和CD
构造域的互相作用研究··············18
2.1.4RBBP1TD，PD和CD
构造域与甲基化氨基酸和组蛋白短肽的互相作用
研究·······································18
2.1.5RBBP1CD的核磁共振实验与数据剖析
···················19
2.1.6RBBP1CD的构造分析··························20
结果·······································20
克隆与表达
·································20
互相作用研究
··································22
2.2.3RBBP1CD的化学位移指认与三维构造
···················26
2.2.4RBBP1CD的溶液构造剖析以及与甲基化组蛋白尾巴联合的构造基础····30
2.2.5RBBP1CD的突变体研究·························32
议论·······································35
第3
章RBBP1TD构造域的构造分析························37
资料与方法····································37
表达纯化··································37
核磁样品··································37
核磁共振实验与数据剖析与构造计算
···················37
目录典范
3.2结果·······································37
3.2.1指认与剖析·································37
3.2.2构造质量··································38
3.2.3构造描绘与剖析·······························40
3.3议论·······································40第四章结论和展望··································444.1主要结论·····································44
4.2工作展望·····································45道谢·········································46参照文件······································47附录组蛋白N端尾巴的修饰位点·························51博士生时期发布的学术论文······························52博士后时期发布的学术论文······························53个人简历······································53永远通信地点·····································54
目录典范
附录典范
缩写表
AD
ARIDdomain
ARID构造域
AS
Angelmansyndromes
安吉尔曼综合症
BMRB
BioMagResBank
生物磁共振数据库
CD
chromobarreldomain
染色质联合桶状构造域
COSY
correlatedspectroscopy
有关谱
CSP
chemicalshiftperturbation
化学位移扰动
DTT
dithiothreitol
二硫苏糖醇
FPLC
fastproteinliquidchromatography
迅速蛋白质液相层析
HDAC
histonedeacetylase
组蛋白去乙酰化酶
HSQC
heteronuclearsinglequantumcoherence
异核单量子有关
NMR
nuclearmagneticresonance
核磁共振
NOE
nuclearOverhausereffect
核Overhauser效应
NOESY
NOEspectroscopy
NOE谱
OD
opticaldensity
光密度
PD
PWWPdomain
PWWP构造域
PWS
Prader-Willi
普拉德威利综合症
RBBP1
Retinoblastoma-bindingprotein1
眼癌蛋白联合蛋白1
RBBP1L1
RBBP1-likeprotein1
RBBP1近似蛋白1
RB
Retinoblastomaprotein
眼癌蛋白
PDB
ProteinDataBank
蛋白质（构造）数据库
RMSD
root-mean-squaredeviation
均方根误差
SDS
sodiumdodecylsulfate
十二烷基硫酸钠
SDS-PAGE
SDSpolyacrylamidegelelectrophoresis
SDS聚丙烯酰氨凝胶电泳
TD
Tudordomain
Tudor构造域
TOCSY
totalcorrelatedspectroscopy
全有关谱
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