Molecular Biology in Medicine 医学分子生物学 许正平. The greatest intellectual revolution...
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Transcript of Molecular Biology in Medicine 医学分子生物学 许正平. The greatest intellectual revolution...
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Molecular Biology in Medicine
医学分子生物学
许正平
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The greatest intellectual revolution of the last 40 years may have taken place in biology. Can anyone be considered educated today who does not understand a little about molecular biology?
─F. H. Westheimer (Harvard University)
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Genetic Information Transfer
遗传信息的传递 Gene Transcription 基因转录 RNA Splicing & Editing RNA 剪切与加工 Protein Synthesis & Processing
蛋白质合成与加工 Regulation of Gene Expression
基因表达的调控 ( 包括 miRNA 、 RNAi)
分子生物学主要内容
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分子生物学主要研究技术 分离、纯化(主要是生物大分子) 克隆、表达 PCR (多聚酶链式反应 ) 凝胶电泳:琼脂糖凝胶电泳; SDS -聚丙烯酰胺凝胶电泳 ( SDS-PAGE );等电聚焦电泳;双向电泳 印迹技术: Southern blotting; Northern blotting;
Western blotting
微阵列技术: genechip, microarray, protein chip
基因操纵技术: Gene-knockout/knockin
RNA interference (RNAi)
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分子生物学主要研究技术 蛋白质相互作用:酵母双杂交、免疫共沉淀( Co-IP )、 pull-down 、 FRET 、表面等离子共振技术 (SPR)
蛋白质鉴定:质谱 研究生物大分子三维结构常用的实验手段: X 射线晶体学、核磁共振、电子显微学、原子力显微镜 以及 X 射线小角散射等。
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定义:从分子水平研究人体在正常和疾病状态下生命活动及其规律的一门科学重点:人体生物大分子和大分子体系的结构、功能、相互作用及其同疾病发生、发展的关系
医学分子生物学
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教材:医学分子生物学(第 3 版)
参考书: Molecular Cell Biology Gene
More Information: Literature Internet
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I. Introduction
The Central Dogma
gene expression: The overall process by whichthe information encoded in a gene is converted into an observable phenotype (most commonly production of a protein).
Prokaryotic vs Eukaryotic
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I. Introduction
How many genes in the human genome?Gene Expression
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I. Introduction
FACT 1: an uniform genome in almost every cell of an organism
FACT 3: the shape and function of each type of cell are different
FACT 2: the proteome in each type of cell is different
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I. Introduction
the actions and properties of each cell type are determined by the proteins it contains
FACT 1: an uniform genome in almost every cell of an organism
transcription of different genes largely determines the actions and properties of cells
FACT 3: the shape and function of each type of cell are different
FACT 2: the proteome in each type of cell is different
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I. Introduction
the types and amounts of the various proteins in a cell
the concentration of mRNA and the frequency at which the mRNA is translated
which genes are transcribed and their rateof transcription in a particular cell type
TRUTH: the gene is differentially expressed
regulation
same genome in all cells of an organism
regulation
regulation
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I. Introduction
Gene Expression Occurs by a Two-Stage Process
Transcription: generates a single-stranded RNA identical in sequence with one of the strands of the duplex DNA Three principal classes of products:
message RNA (mRNA)transfer RNA (tRNA)ribosomal RNA (rRNA)
Principle: complementary base pairing
Translation: converts the nucleotide sequence of an RNA into the sequence of amino acids comprising a protein
each mRNA contains at least one coding region that is related to a protein sequence
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II. Transcription
DNA (gene)
RNA polymerase
Regulatory Proteins
Key Players
promoter
A
startpoint terminator
Transcription Unit
template
upstream downstream
enhancer
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II. Transcription
Primary transcript is the original unmodified RNA product correspondingto a transcription unit.
Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription.
RNA polymerases are enzymes that synthesize RNA using a DNA template(formally described as DNA-dependent RNA polymerases).
Terminator is a sequence of DNA, represented at the end of the transcript,that causes RNA polymerase to terminate transcription.
Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene.
Key Terms
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Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase:
RNA polymerase I:
RNA polymerase II:
RNA polymerase III:
RNA Polymerase
II. Transcription
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Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase:
RNA polymerase I: rRNA; resides in the nucleolus
RNA polymerase II: mRNA, snRNA; locates in the nucleoplasm
RNA polymerase III: tRNA and other small RNAs; nucleoplasm
II. Transcription
RNA Polymerase
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The promoters for RNA polymerases I and II are (mostly) upstreamof the startpoint, but some promoters for RNA polymerase III lie downstream of the startpoint.
Each promoter contains characteristic sets of short conserved sequences that are recognized by the appropriate class of factors.
RNA polymerases I and III each recognize a relatively restricted setof promoters, and rely upon a small number of accessory factors. Promoters utilized by RNA polymerase II show more variation in sequence, and are modular in design.
Promoter
II. Transcription
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Short sequence elements (cis-acting elements): bind by accessory factors (transcription factors)
The regulatory region might exist in the promoters of certain eukaryotic genes.
Location: usually upstream and in the vicinity of the startpoint.
These sites usually are spread out over a region of >200 bp. common: used constitutivelyspecific: usage is regulated; define a particular class of genes
These sites are organized in different combinations
Cis-acting Element
II. Transcription
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Enhancer element is a cis-acting sequence that increases the
utilization of (some) eukaryotic promoters. The components of an enhancer resemble those of the promoter.
Involve in initiation, but far from startpoint. Are targets for tissue-specific or temporal regulation. Function in either orientation and in any location (upstream or
downstream) relative to the promoter.
Enhancer
two characteristics:1. the position of the enhancer need not be
fixed.2. it can function in either orientation.
II. Transcription
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promoter enhancer
position fixed variable
action direction one way either orientation
the density of regulatory elements sparse Heavy (closed packed)
redundancy in function no yes
cooperativity between the binding of factors
sequential great
The Difference between Promoter and Enhancer
The distinction between promoters and enhancers is operational, rather than imply a fundamental difference in mechanism
II. Transcription
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Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements
(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancersas well as a TATA box or other promoter element. Enhancers may be either upstream or downstream and as far away as 50 kb from the transcription start site. In some cases, promoter-proximal elements occur downstream from the start site as well. (b) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site.
II. Transcription
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Fact: Regulatory elements in eukaryotic DNA often are many kilobases from start sites
Finding Regulatory Element in Eukaryotic DNA
II. Transcription
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Transcription Factor
Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor.
binds to DNA (trans-acting factor): recognize cis-acting elements
interacts with other protein: recognize RNA pol, or another factor
The common mode of regulation of eukaryotic transcription is positive: a transcription factor is provided under tissue-specific control to activate a promoter or set of promoters that contain a common target sequence. Regulation by specific repression of a target promoter is less common.
II. Transcription
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Accessory factors are needed for initiation, principally
responsible for recognizing the promoter.
Interaction with DNA, RNA polymerase, and/or another
factors.
Three groups:
1. General factors
2. Upstream factors
3. Inducible factors
Another name: accessory
II. Transcription
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general factors: required for the mechanics of initiating RNA synthesis at all promoters; form a complex surrounding the startpoint with RNA pol, and determine the site of initiation.
basal transcription apparatus (pol + GF)
upstream factors: DNA-binding proteins that recognize specific short consensus elements located upstream of the startpoint. not regulated; ubiquitous; act upon any promoter that contains the appropriate binding site on DNA.
inducible factors: function in the same general way as the upstream factors. have a regulatory role: control transcription patterns in time and space
Accessory Factors
II. Transcription
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II. Transcription
Fou
r S
tage
s in
T
ran
scri
pti
on
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1. On the genome Which gene(s) to be transcribed? Basic events: Protein binding and/or modification 2. On a specific gene If the gene can be transcribed successfully?
3. On a transcript If the transcript could be correctly spliced? If the transcript could be correctly edited?
Regulation Levels
Key determinant: Cell Signaling!
III. Regulation of transcription
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Potential regulation points
Activation of gene structure↓
Initiation of transcription↓
Processing the transcript↓
Termination of transcription↓
Transport to cytoplasm
the overwhelming majority of regulatory events occur at the initiation of transcription
III. Regulation of transcription
5 potential control points:
“Active” Structure
Major Control Point
Alternative Splicing
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Regulatory Proteins
the overwhelming majority of regulatory events occur at the initiation of transcription
Key player: regulatory transcription factors
Two questions:1. How does the transcription factor identify its group of target genes?2. How is the activity of the transcription factor itself regulated in response to intrinsic or extrinsic signals?
III. Regulation of transcription
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Answer to question 1
The genes share common response element
Structure feature: contain short consensus sequence
Examples:
HSE: heat shock response element; recognized by HSTF
GRE: glucocorticoid response element
SRE: serum response element
MRE: metal response element
III. Regulation of transcription
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Regulatory region in MT gene
BLE: basal level element; TRE: TPA response element
General Principle: any one of several different elements, located in either anenhancer or promoter, can independently activate the gene.
III. Regulation of transcription
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Answer to question 2
Signal transduction
Key events:
1. Protein synthesis
2. Protein modification
3. Ligand binding
4. Protein cleavage
5. Inhibitor release
6. Mutation
III. Regulation of transcription
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The activity of a regulatory transcription factor may be controlled by synthesis of protein, covalent modificationof protein, ligand binding, or binding of inhibitors that sequester the protein or affectits ability to bind to DNA.
Reg
ula
tion
Mod
es o
f T
ran
scri
pti
on
Fac
tor
mutations of the transcriptionfactors give rise to factors thatinappropriately activate, or prevent activation, of
transcription
III. Regulation of transcription
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Eukaryotic transcriptional control operates at three levelsduring the stage of initiation
1. changes in chromatin structure directed by activators and repressors
2. modulation of the levels of activators and repressors(gene expression)
3. change the activities of activators and repressors
III. Regulation of transcription
Gene differential expression
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IV. RNA Processing
INTRODUCTION
Facts:
1. Genes are interrupted, and mRNAs are uninterrupted
2. The primary transcript has the same organization as the gene
3. mRNA has 5’ cap and 3’ poly(A) tail
4. Heterogeneous nuclear RNAs (hnRNA) exist in the nucleus
5. RNA contains rare bases
Mechanism:
RNA splicing: remove intron
RNA modification: 5’ capping, 3’ polyadenylation, base modification
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INTRODUCTION
The initial primary transcript synthesized by RNA polymerase IIundergoes several processing steps before a functional mRNA is produced:
5’ capping 3’ cleavage/polyadenylation RNA splicing
RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exonsare connected into a continuous mRNA.
IV. RNA Processing
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Overview of mRNA Processing in Eukaryotes
The poly(A) tail: ~250 A in mammals, ~150 in insects, ~100 in yeasts. For short primary transcripts with few introns, polyadenylation, cleavage, and splicing usually follows termination. For large genes with multiple introns, introns often are spliced out of the nascentRNA before transcription of the gene is complete.
IV. RNA Processing
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Th
e sp
lice
osom
al s
pli
cin
g cy
cle
The splicing snRNPs associate with the pre-mRNA and with eachother in an ordered sequence to form spliceosome
ATP is needed to provide theenergy necessary for rearrangements of the spliceosome structure
IV. RNA Processing
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Alternative splicing
Mechanisms:• use of different startpoints or termination sequences• a single primary transcript is spliced in more than one
way, and internal exons are substituted, added, or deleted
Definition: a single gene gives rise to more than one mRNA sequence
Key:what controls the use of such alternative pathways?
IV. RNA Processing
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Alternative splicing
Mechanisms:• use of different startpoints or termination sequences• a single primary transcript is spliced in more than one
way, and internal exons are substituted, added, or deleted
Definition: a single gene gives rise to more than one mRNA sequence
Key:what controls the use of such alternative pathways?Protein(s)!
IV. RNA Processing
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The Troponin (肌钙蛋白) T (muscle protein) pre-mRNA is alternatively spliced to give rise to
64 different isoforms of the protein
Constitutively spliced exons (exons 1-3, 9-15, and 18)
Mutually exclusive exons (exons 16 and 17)
Alternatively spliced exons (exons 4-8)
Exons 4-8 are spliced in every possible waygiving rise to 32 different possibilities
Exons 16 and 17, which are mutually exclusive,double the possibilities; hence 64 isoforms
IV. RNA Processing
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Trans-(intermolecular) splicing
Splicing is usually cis-reaction (intramolecular), but trans-(intermolecular) splicing have been found (very rare). These reactions probably occur by splicesome formation with the appropriate site sequences on each molecule.
trypanosomes and euglenoids: all the mRNAsCaenorhabditis elegans: 10-15% of the mRNAsHuman?
IV. RNA Processing
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Initiation of Protein Synthesis
V. Initiation of Protein Synthesis
Critical event:begin protein synthesis at the start codon, thereby setting the stagefor the correct in-frame translation of the entire mRNA.
Main mechanisms:Base pairing between mRNA and rRNABase pairing between mRNA and tRNAMet-tRNAi
Met can only bind at the P site to begin synthesis
Participants: Met-tRNAi
Met
mRNA IFs small subunit large subunit
Protein translation
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Two types of methionine tRNA are found in all cells
same aminoacyl-tRNA synthetase (MetRS) charges both tRNAs with methionine
V. Initiation of Protein Synthesis
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Eukaryotic initiation of protein synthesis
V. Initiation of Protein Synthesis
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Model of protein synthesis on circular polysomes and recycling of ribosomal subunits
PABI and eIF4 (4G and 4E) can interact on mRNA to circularize the molecule
V. Initiation of Protein Synthesis
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The nascent polypeptide chain must undergo folding and, in many cases, chemical modification and cleavage to generate the final protein
Folding:Theoretically: any polypeptide chain containing n residues could, in principle, fold into 8n conformations. Fact: adopt a single conformation (native state)
a single, energetically favorable conformation Mechanism: the amino acid sequence provides the information for protein folding
Protein Maturation
Modification: N terminal C terminal Certain sites btw N and C terminus
VI. Protein Processing
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Nearly every protein in a cell is chemically altered after its synthesis in a ribosome, thus alter its activity, life span, or cellular location of proteins,
depending on the nature of the alteration.
Two categories:chemical modification involves the linkage of a chemical group to the terminal amino or carboxyl groups or to reactive groups in the side chains of internal residues may be reversibleProcessing involves the removal of peptide segments and generally is irreversible
Protein Alteration
VI. Protein Processing
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The internal residues in proteins can be modified by attachment of a variety ofchemical groups to their side chains:phosphorylation (Ser, Thr, Tyr) glycosylation (Asp, Ser, Thr)ubiquitinationothers
Examples of modified internal residuesproduced by hydroxylation, methylation, and carboxylation
Protein Modification
VI. Protein Processing
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Protein Cleavage
most common form:
residues are removed from the C- or N-terminus of a polypeptide
by cleavage of the peptide bond in a reaction catalyzed by
proteases.
Proteolytic cleavage is a common mechanism of activation or
inactivation
Proteolysis also generates active peptide hormones
EGF; insulin
VI. Protein Processing
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Protein Degradation
Two Pathways
extracellular: digestive proteases
intracellular
lysosomes
cytosolic mechanisms
The ubiquitin-mediated pathway is the best-understood cytosolic pathway.In ubiquitinating enzyme complex, different conjugating enzymes recognize different degradation signals in target proteins.
ubiquitin-conjugating enzyme E1: Arg-X-X-Leu-Gly-X-Ile-Gly-Asxcertain residues at the N-terminus favor rapid ubiquitination
VI. Protein Processing
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基因突变、多态性:个体易感性、疾病发生表观遗传修饰:功能改变蛋白质量 / 结构 / 构象 / 功能改变:非正常功能、疾病调控异常:非正常功能、疾病
分子生物学与医学
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医学分子生物学的应用
阐明生理 / 病理现象的分子机制 遗传病诊断(基因诊断) 发现疾病的生物标志物,为诊断服务 疾病的生物治疗 药物研发与评价(药物基因组学) 个性化医疗
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Tumor Angiogenesisand
The Role of Angiogenin
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Part I Tumor Angiogenesis
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Angiogenesis
Angiogenesis: the outgrowth of new blood vessels from pre-existing vessels
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Normal angiogenesis Tumor angiogenesis
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InhibitorsInhibitorsActivatorsActivators
FolkmFolkm
anan
The turn on and off of angiogenesis
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The process of angiogenesis
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The process of angiogenesis
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Tumors can be cured through ‘starving to death’. Avastin (VEGF monoclonal antibody) was approved for combination use with standard chemotherapy for metastatic colon cancer and non-small cell lung cancer in 2004.
Anti-angiogenesis & anti-tumor therapy
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Anti-angiogenesis: main target
activation of target cell:ligand-receptor bindingsignalling
cell migrationMMPs
cell adhesion
Ideal target: a specific event in tumor angiogenesis
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Anti-angiogenesis: reagent
protein or antibodye.g. avastin
peptide
small molecule
Ideal reagent: no or negligible side-effect
http://www.cancer.gov/cancertopics/factsheet/Therapy/angiogenesis-inhibitors
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Anti-angiogenesis: main problem
multi-factor
multi-pathway
side-effect
solution: combined action!
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Thalidomide: a scandal
Thalidomide: chiefly sold and prescribed to
pregnant women from 1957 to 1961 in almost
50 countries, as an antiemetic to combat morning
sickness and as an aid to help them sleep.
catastrophic results: severe malformities, including
phocomelia( 海豹肢症 )
Reason: the developing fetus will not form blood
vessels properly and thereby stop the proper
development of fetal limbs and circulatory systems.
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Part II The role of angiogenin
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Angiogenin (ANG) is one of the major angiogenesis activators Angiogenin (ANG) is one of the major angiogenesis activators
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N
C
ANG was originally isolated from the cond
itioned medium of cultured HT-29 human col
on adenocarcinoma cells based solely on its
angiogenic activity.
Mature ANG is a basic, single-chain protei
n containing 123 amino acids with a molecul
ar weight of about 14,400 Da.
ANG gene is present as one copy per hap
loid genome, and localizes on chromosome
14q11.
ANG is a member of ribonuclease superfa
mily.
A brief introduction to angiogenin (ANG)
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Angiogenin as a potential drug target
• The expression of angiogenin is up-regulated in various ty
pes of human cancers.
• Many molecules targeting ANG, such as ANG monoclonal
antibodies, have been reported to exert antitumor effects.
• However, they all antagonize angiogenin itself, and may h
ave significant side effect.
• An ideal angiogenin-oriented drug could be made possible
only after fully elucidating the mechanism of action of ang
iogenin and identify a disease-specific process.
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Mechanisms of Action of Angiogenin
• Angiogenin induces angiogenesis mainly through:– binding to membrane actin and then inducing basem
ent membrane degradation– binding to a putative 170-kDa protein and subseque
ntly transducting signal into cytoplasm – translocating into the nucleus of target cells directly
and then enhancing rRNA transcription – exerting its ribonucleolytic activity
• Angiogenin can also translocate into the nucleus of cancer cells and induces the corresponding cell proliferation
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?
?
?
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Research Interest:
Elucidating the mechanism of action of angiogenin
1. Angiogenin-interacting proteins
2. Angiogenin-stimulating miRNAs
Developing anti-angiogenesis and anti-tumor molec
ules
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• Since protein interactions are critical in every biologica
l process, interactions between ANG and other proteins
should mediate or modulate a series of biological activi
ties in ANG-induced angiogenesis and tumor cell growt
h.
• The study of angiogenin-interacting proteins will help t
o understand the molecular mechanism of angiogenin-i
nduced angiogenesis and tumorigenesis.
• The study of ANG PPIs may also provide potential targ
ets for anti-angiogenesis therapy.
Why study ANG-interacting proteins?
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Yeast Two-hybrid
Co-IP
Approaches employed to study ANG-interacting proteins?
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Identify ANG-interacting proteins using yeast two hybrid system
Yeast two-hybrid screening
Interaction confirmation
Mapping the binding sitesExploring the significance of the interaction
Peptide synthesis &Interaction interference
Drug design
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Basis of the Two-Hybrid System.(GIBCO BRL® instruction manual)
Principle of yeast two-hybrid
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Functional classifications of ANG-binding proteins
ANG-interacting proteins Functional classifications
Laminin
Extracellular matrix proteinsFibronectin
Fibulin1,2,3,4
Latent transforming growth factor-beta binding protein3,4 (LTBP3,4)
Progranulin
Regulatory factors
Follistatin
Sprouty
VE-statin
Chordin
Synovial apoptosis inhibitor 1
Phospholipid Scramblase 1
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ANG-interacting proteins Functional classifications
Alpha actinin 2 (ACTN2) Cytoskeleton protein
A Disintegrin And Metalloproteinase protein33 (ADAM33)
Membrane protein
Interferon, alpha-inducible Protein 27 Others
Sarcolemma Associated Protein
FLJ25471, FLJ00221
21 proteins were identified as potential ANG-binding proteins using Y-2-H method.
Functional classifications of ANG-binding proteins
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Verification of the potential aniogenin-interacting proteins
reconfirmation in the yeast system
pull-down
in vitro Co-IP
in vivo Co-IP
FRET
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a-Actinin-2 (ACTN2), a cytoskeletal protein, binds to angiogenin
ANG ACTN2
- -
- +
+ -
+ +
Identification of ACTN2 as a potential ANG-binding protein in Y-2-H analysis
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Detecting the interaction between ACTN2 and ANG using His pull-down
experiment
ACTN
ACTN
His-ACTN2 - + +
WB: ANG
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Detecting the interaction between ACTN2 and ANG using Co-Immunoprecipitat
ion experiment
Co-immunoprecipitation experiment
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Detecting the interaction between ACTN2 and ANG in vivo using FRET
experiment
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Mapping the ANG-binding sites on ACTN2
aa383-632 on ACTN2 was required for ANG binding
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Crystal Structure Of The Rod Domain Of Alpha-Actinin
aa383-632 (yellow) on ACTN2 was required for ANG binding
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Mapping the ACTN2-binding sites on ANG
aa83-123 on ANG was required for ACTN2 binding
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R101 on ANG plays an important role in ANG-ACTN2 interaction
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R101
R101 on ANG plays an important role in ANG-ACTN2 interaction
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Confirmation of the interaction between Phospholipid Scramblase 1 (PLSCR1) and ANG
PLSCR1 interacts with ANG in yeast PLSCR1 interacts with ANG in vitro
Yeast two-hybrid analysis GST pull-down experiment
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Confirmation of the interaction between PLSCR1 and ANG
PLSCR1 interacts with ANG in vivo
PLSCR1 co-localizes with ANG in the nucleolous
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PLSCR1 enhances rRNA transcription in HeLa cells
Over-expression of PLSCR1 increases45S rRNA level
Down-regulation of PLSCR1 decreases 45S rRNA level
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U6 Ai Ai+ANG
PLSCR1 enhances rRNA transcription through angiogenin
U6: ControlAi: ANGi
ANG expression was down-regulated in ANG RNAigroup and can be recovered with ANG addition
PLSCR1 enhances rRNA transcription only in the presence of ANG
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FS interacts with ANG in yeastFS interacts with ANG in yeast
FS interacts with ANG directly in vitroFS interacts with ANG directly in vitro FS interacts with ANG in HeLa cellsFS interacts with ANG in HeLa cells
Identification of the interaction between follistatin (FS) and angiogenin
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Follistatin binds to angiogenin through FS1 and FS2 domains
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FS inhibits rRNA transcription in HeLa cells
FS localized in the nucleolous of HeLa cells
FS
Nucleolin
Merge
Control FS over-expression
Over-expression of FS decreases45S rRNA level
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Follistatin inhibits angiogenin-induced ABE transcription activity
ABE: angiogenin-binding element. It plays an important role in angiogenin-stimulated rRNA transcription.
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Principle of Co-Immunoprecipitation
Western Blot or MS
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Screen ANG-binding proteins under physiological
circumstance
Flow chart of Co-IP experiment
Cell lysis
ANG+ ANG-
Immunoprecipitation with ANG Abs
MS analysis
Silver staining, looking for differential band
Electrophoresis
Western Blot to verify the binding of ANG and candidate proteins Silver staining of ANG-immunoprecipitates
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ANG-interacting proteins Functional classifications
Far upstream element (FUSE) binding protein
Transcription
Y box binding protein 2
Damage-specific DNA binding protein 2
Ubiquitin-like, containing PHD and RING finger domains, 1
SUB1 homolog (S. cerevisiae)
Eukaryotic translation initiation factor 4A, isoform 2
Translation and regulation of protein metabolic process
Tu translation elongation factor, mitochondrial
Heat shock 70kDa protein 9
Functional classifications of ANG-binding proteins
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Functional classifications of ANG-binding proteins
Actin, beta
Cytoskeleton, cell motility and junctions
Myosin, heavy chain 9, non-muscle
Alpha actinin 4 (ACTN4)
IQ motif containing GTPase activating protein 1
Spectrin, alpha, erythrocytic 1
Ribonuclease/angiogenin inhibitor 1
Others
Argininosuccinate synthetase 1
Lectin, galactoside-binding, soluble, 12
EF-hand domain family, member D2
T cell receptor beta variable 20-1
Cold inducible RNA binding protein
19 proteins were identified as potential ANG-binding proteins using Co-IP coupled with MS method.
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conclusion
• We have identified a bunch of angiogenin-binding partner
s using Y-2-H and Co-IP method.
• Our results showed that PLSCR1 increased ANG-stimulat
ed rRNA transcription while FS acted as a negative regula
tor.
• The significances of the interactions between ANG and ot
her proteins are under study, especially the extracellular
matrix proteins and those related to cell motilities.
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PLSCR1FSGRN
Cytoskeleton and cell motility?
?
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Thank you for your attention!