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1、1,1. 现有“生物信息学”的限制： “有眼不识泰山” 第一外显子 识别能力 外显子内含子 识别能力 2. 一个基因：不同剪切体 Splicing form (Isoform) 3. 一个基因 蛋白质 切割： 蛋白质 1 肽段,人基因组计划已经给了我们多少？,人基因有多少个？,10万 3万？,2,4. 同一个蛋白质 细胞内的不同 “定位” 不同功能！ 5. 98 DNA序列：功能不明 ! （95）,细胞核,细胞质,线粒体,细胞膜,蛋白质移位 Translocation,3,E1 b E1 a Exon 2 Exon 3,p16 Ink 4a,P14 ARF (human) P19 ARF (m
2、ouse),Cross Talk betweren Oncogenes & tumor suppressors,Oncogenic Signals Ras, c-myc, E2F,p16 Ink 4a,Rb,on,Cell cycle arrest,P14 ARF (Block MDM-2 / p53 binding),p53 on,DNA Damage,一个基因可编编码两种完全不同的蛋白质,4,功能基因组学 Functional Genomics,阐明基因（蛋白质）之间的相互作用 与生命活动的关系 与疾病的关系 与环境、药物的关系 基因组测序只是基因组研究的起点 “功能基因组学”的含义被“局
3、限地”误解 “基因表达谱”的误解或局限性 cDNA芯片：mRNA表达蛋白质表达 蛋白质芯片：只知道蛋白质的表达与存在 并不知道蛋白质的功能状态,问题：,在基因组的水平,5,1. 糖基化 (1) 膜表面蛋白：影响细胞识别，细胞细胞相互关系 (2) 细胞表面抗原： (3) 细胞内蛋白： 特定糖基化与蛋白质叠成为高级结构有关 成熟蛋白质不成熟蛋白质的“质控”表志 2. 乙酰化： 组蛋白：与DNA结合能力、复制、转录 其它蛋白质 （如p53） 3. 磷酸化： 4. 蛋白质切割成片段：一个蛋白质多种功能。,6,细胞分子细胞生物学重要问题,过度增殖： 恶性肿瘤 高血压（内皮细胞、平滑肌细胞） 细胞凋亡：
4、神经退行行病变 早老性痴呆 帕金森氏病 “ To be or not to be, thats a question” HamletShakespear,细胞 增殖,细胞 凋亡,生 死,7,（抗凋亡） Antiapoptotic,（促进凋亡）,M,Check Points of Cell Cycle G1 S check point G2 M check point M: Mitotic Spindle checkpoint,8,分子 细胞 组织 机体,误 区：相互分离。每种疾病均集中于反映出疾病的细胞 肿 瘤：集中在癌细胞 误 区：忽视癌细胞以外的细胞 心血管疾病： 心脏病 心肌细胞 心肌间
5、质血管内皮细胞 高血压 内皮细胞 内皮细胞间质细胞炎症细胞 平滑肌细胞 神经性疾病： 神经原细胞 神经原 胶质细胞 间质 内皮细胞（高血压脑病）,9,间质细胞 血管内皮细胞 淋巴管内皮细胞 淋巴细胞 巨噬细胞 触突状细胞 Dendritic cell,Cytokine (细胞因子) Cytokine Receptor 细胞因子受体 Chemokine(趋化因子) Chemokine Receptor趋化因子受体,恶性肿瘤,除肿瘤细胞外，还有重要的细胞：,10,CANCER:,Multiple Cross-talk among Cancer, stromal and endothelial Ce
6、lls,Cancer cells,Stromal cells,Endothelial cells,Intercellular Cross talk,?,Cancer cells,Matrix (ECM) Endothelial cells Pericytes Stromal cells,11,Stroma:,1. Fibroblasts in tumor: “Activated fibroblast” GF GF R activated 2. Genetic alteration: Juvenile polyposis syndrome (JPS), Cowen syndrome etc Do
7、minant hereditary Stromal cells abnormality, Hamartoma High risk for colon cancer PTEN and others “Landscaper” “Stroma abnormal microenviroment” “Microenviroment of tumor-host interface ”,12,ANGIOGENESIS,Two Processes Endothelial Cells (EC) growth migration Vessel formation Tube formation extension
8、Other cells: Fibroblasts etc,13,ANGIOGENESIS / CANCER INVASION,EC Growth control & Vessel formation,Hypoxia Activating hypoxia responses genes ( p53 ) Acidosis VEGF-A ( Cancer. EC ),14,Protease / matrix / Cancer cell invasion / Angiogenesis,Proteases: Degradation of ECM and Cell Associated Proteins
9、MMPs ( Marix metallo proteinases), membrane - anchored MMP 2, MMP 9, (Cancer Cell) Soluble MMP (fibro, EC) Adamalysin related membrane proteinases BMP 1 metalloproteinases Tissue serine proteinases tPA UK Thrombin Plasmin uPA ( EC, fibro. ) uPAR ( Cancer ),Inhibitors PAI TIMPs ( Tissue inhibitor of
10、MMPs) PEX ( Degradation product of ECM by MMP 2 ),15,Proteases further create Plasminogen Angiostatin (38KD) EC ATP synthase ( I ) Procollagen XXIII Endostatin (20KD) Prolaction 16KD prolactin ECM PEX EC integrin ab3 ( I ) RGD fragment Cancer FAK (S) Troponin Tn-1 EC ATP Synthase ( I ) (Human cartil
11、age),?,Inhibitors of Angiogenesis Stimulators of Cancer Invasion,( I ),ANGIOGENESIS / CANCER INVASION,16,EC Receptors: ( EC and Cancer Cell ) VEGF R 1 (Flt-1) VEGF R 2 (Flk-1, KDR) VEGF A-D PLGF VEGF R 3 (Flt-4) VEGF C, D TIE 1 ? TIE 2 Ang 1, 2 3? 4?,* Integrin ab3 * ECM PEX ab5 (Laminin, tenasin) C
12、D 36 Thrombospondin (ECM Glycoprotein) cell adhesion, motility & growth metallospondin,Vascular Tubule formation,* Eph B1 Recognizing some ligands or partners NHE 3 ? ARNT ? EPA-1 ?,Tenasoin: new ECM.,ANGIOGENESIS / CANCER INVASION,17,VEGF Rs,VEGF R1 VEGF R2 VEGF R3 Neurophilin 1 Neurophilin 2 (flt-
13、1) (flk-1 KDR) (flt 4),Ligands:,18,EC Growth Factors:,Specific GFs: VEGF A-D VEGF R2 (KDR. Flk-1) PLGF VEGF R1 (flt-1),Non-specific GFs (Local): Some of them: autocrine (EC, Cancer Fibro, ) TGFa EGF R TGFb ( fibro) TGFb R ( Cancer ) TGF b / ECM TGF b released aFGF bFGF FGF R PDGF PDGF R HGF Met,ANGI
14、OGENESIS / CANCER INVASION,Protease,19,Chemokines,CXC chemokines Recruit monocytes (Tumor cell origin) leukocytes or others CXC L12 CXC L-12 enriched tissue CCR4 metastasis Pseudopodia, migration, penetration of ECM, homing,Induce TNFa,Cytokines related to EC or Capillary formation,IFN a Downregulat
15、e VEGF production bFGF IFNg Inhibit angiogenesis IL-12 Upregulate IFN-g & IP-10, inhibiting angiogenesis,ANGIOGENESIS / CANCER INVASION,20,Interaction between cancer cells & EC,Hypoxia Proteases Degradation of ECM VEGFs Angs TGEa bFGF PDGF ? Chemokines,cancer,EC,ANGIOGENESIS / CANCER INVASION,Metast
16、asis,21,Enigmas,1. Tumor / endothelial cells Cross-talk 2. Tumor cells endothelia like cells. Morphological evidence: yes Biochemical: ? 3. Stromal cells / tumor cells Stromal cells / endothelial cells 4. Genetic alteration of tumor vascular endothelial cells ? Yes or not? 5. Genetic alteration of t
17、umor stromal cells,Cross-talk,22,APOPTOSIS,1. Death signals: TNFa family 2. Death Receptors: TNF Receptor family, Decoy Receptor 3. Adaptor molecules: Interact with receptor Caspase (Protocaspase) 4. Proto caspases: Activated Caspases 5. Effector molecules: Other Proteases, DNA degradation enzymes 6
18、. Inhibitors: For Caspases Adaptors Others,New Progress,Molecules inside mitochondria release into cytoplasm (Procaspases, cytochrome C ctc.) Nuclear / Cytoplamic molecules translocate to MT membrane,23,Mitochondrion / Cytoplasm,Caspases Other Signals,In intermembrane space Protocaspase 2 9 3 Latent
19、 AIF Cytochrcme C Smac / DIABLO,Life,Other targest,Mitochondrion,Protocaspase-2 Protocaspase-3 Protocaspase-9 Apaf 1 Cytochrome c AIF Others ? (Smac / DIABLO),Life or Death ?,Alteration of Molecular Compartmentation (Mitochondrion versus Cytoplasm),Other Targets,Intermembrane Space,AIF (Apoptosis in
20、ducing factor) Science, 397: 3879 1999 Smac (Second MT-derived activator of caspase) Or DIABLO (Direct IAP-binding protein at Low pI),Bcl 2 guard ?,Death,24,Mitochondria-death Signal Integrators,C. Bremer & G. Koemer (Science. Aug. 18.289: 1150, 2000),Translocate to mitochondrin Stimulation / induct
21、ion Inhibition / block translocation (Keep in cytosol),PTPC: Permeability Transition Pore Complex (Bax etc. ) Bcl2 / Bax / Bid permeabilize the outer MT membrane upon interaction with PTPC Bid may be independent JNK: (SAPK, stress activating protein kinase) - inactivate Bcl XL p53: Translocated to M
22、T & interacted with hsp 70 PKC: Ttranslocated to MT,25,Enigmas in Apoptosis,Translocation of nuclear or cytosolic protein molecules into mitochondria,Phosphorylation / dephosphorylation Translocator Protein / protein binding conformational change Binding to mitochondria surface membane protein,Trans
23、location or release of proteins or other molecules from mitochordria to cytoplasm,Permeability transit pore complex Voltage depedent anion channel (VDAC),Apoptosis induced by paradoxical signals,Growth factor / receptors Protooncogenes: Ras, c-myc etc.,26,HOT SPOTS OF SIGNALING,20% of 32,000 human c
24、oding gene: Signal transduction Protein Kinase 520 Protein Phosphatase 130 Dominant Oncogenes 100 PK Tumor Suppressor 30 PK Known PTK PTK 90 (May, 2001) RPTK 58 (20 families) Non-receptor, PTK 32 ( 10 families) Cytoplasmic,27,Human RPTK,28,c-src: Truncated mutation of C to Tyr 530 autophosphorylatio
25、n related to STAT-3 in colon cancer c-abl: CS 9 (9:22) abl / BCR in ph1 CS DNA damage induced apoptosis. ATM c-abl bind Rb at G0/G1 (released after RB phosph. of abl prevent DNA-damage induced abl phosph. PDGF Induce cell motility & adhesion (c-abl involved in) BCR / abl Cyteplasm X nucleus,Human Cy
26、toplasmic PTK,29,1. Class I p110 (catalytic) Adaptor / regulator IA: RPTK activated IB: hetero-trimeric G protein coupled Receptor activated. Class II p1(3)k 2. Substrate: ptd Ins (4.5) P2 ptd Ins (3.4.5) p3 Some P1(3k) (I & III): + Ser / thr K 3. Isoform a, b, d of p110. Subunit p85a, p85b, p55g of
27、 adaptor 4. Binding of protein to lipid IA FVVE domain ptd Ins (3) P PH(pleckstrin homology) domain PH(+) protein: 3-phospho-inositide dependent K (PDK 1) (ser/thr PK) Akt (PKB),PI(3)K / Akt & mTOR / p7056k (rapamycin target),PI(3k):,hetero dimer,IA,ptd Ins(3,4) P2 ptd Ins(3,4,5)P3,30,Akt ( PKB ),Ak
28、t: v- Akt homolog PKB a, b, g isoforms N PH domain Central kinase domain activation loop: Thr. 308 C Regulatory site: Ser 473 PDK- 1 (3-phospho-inositide dependent Kinase 1) : Thr 308 Akt kinase C PH domain 10 fold binding affinity to lipid membrane (constitutively binding) PDK-2: Ser 473 Akt,31,Sub
29、strate of Akt-1,32,Substrate of Akt: 13: RXRXXS/T motif (this motif:also for MAPKAPK-1 & p7056K),Two major class,33,RPTK activation P1(3)k Ptd Ins (3,4,5) P3 Ptd Ins(3,4)P2 Interact with AKT Akt / PDK1 Akt translocatin to membrane, Thr 308 (loop) Ser 473 (Reg. Site) (PDK2 ?),PDK1/Akt activation:,Akt
30、,translocate to nuclei,AKT,PDK1 also PKC isoforms, serum and glucoorticoid induced K (GSK) PKC related K (PRK) p7056k p21 activated PK (PAK),34,PTEN,LOF of PTEN Akt also p27 LOF in glioblastoma, germ cell cancer, breast cancer Cancer & Pl (3)K IA / Akt Akt expression : pancreatic, ovarian cancer Akt
31、 / p65 (mutant of p85 a Pl(3)k) p110 (Pl(3)K) amplification (ovarian cancer) Pl(3)K IB (p110 g ) LOF: colon cancer (Bcl2, CDK. Cyclin D ),(3-phosphoinositide phosphatase, TS) ptd Ins (3,4,5) P3 ptd (3,4) P2,35,mTOR ( Mammalian Target of Rapamycin ),1. Conserved family: Yeast: TOR1, TOR2, MEC1, TEL1,
32、 Rad 3 Drosophila: MEC41 Mammal: mTOR, ATM, ATR ( ATM related ) TRAPP ( transformation / transcriptional domain-associated P ) 2. mTOR = FK506 binding P (FKBP) (human) Rapamycin associated P (FRAP human) Rapamycin and FKBP12 target-1 (rat) 3. mTOR C terminal K domain (homologous to kinase domain of
33、Pl(3)K and Pl(4)K ser / the K activity only mTOR c-myc (inhibited by mTOR inhibitor rapamycin) stat 3 ( ser 727) PKC ; PKC 4E-BP dissociation & release of elf-4E cap dependent initiation translation of 5-UTR,36,P1 (3) K dependent cyelin D3 Phosphorylation of Rb and p107 E2F Involved in P1 (3) K and
34、Akt tumorigenesis Dependent on Phosphorylation & activation of p70S6K,Ribosomal S6 Kinase S6K1, S6K2,Regulator of cell growth & protein translation p70S6K short isoform of S6K 1 Cytoplasic in large part Activation is blocked by rapamycin Mediate mTOR effect on protein-translation Phosphorylation of
35、S6 subunit (40s rs protein) S6 translation of 5 ter. Pyr-rich tract (5-TOP mRNA),Also involved in cell cycle,37,Wnt Pathway,Wnt Wnt 1 = ( int-1, mouse MMTV integration activated gene) Porcupine: A gene for wnt secretion Polytopic membrane protein Related to acyl transferase Frizzled ( Fz ) Wnt Recep
36、tor Dally: Co-receptor of Wnt Glypican-type heparan sulfate proteoglycan Glycosyl-Ptd Ins moiety membrane b-catenin: in absence of Wnt Destabilized by a protein complex Dishevelled (Dsh) activated by Wnt-Fz binding Axin APC GSK-3b (Glycogen synthase kinase) GSK - 3b - b-catenin - b catenin degradati
37、on Wnt Fz binding Dsh GSK - 3b b-catenin stabilized enter nucleus Nuclear transcription factor: Tcf / LEF: activated by b-catenin,38,Wnt Pathway,TCF / LEF,APC,GSK 3b,TCF / LEF,b-catenin,LRP,Fz,39,Hedgehog Pothway,Hedgehog: Embryonic development inducer Hh, hh: Humna Shh (Sonic hh) Ihh (Indian hh) Dh
38、h (Desert hh) 19KD protein C Cholesterol ester N Palmitoylation (after cholesterol addition, enhancing signaling) Secretion of hh: 2 genes: Dispatched related to hh R Patched hh release Toutvelu: enzyme, heparin-SO4 synthesis for transport and response of hh Receceptor of hh Patched (Ptc): Ts, 12 Sp
39、an-transmembrane Proton-driven lipid transportor Smoothened (Smo): Onc, 7-transmembrane p ( Fz like ),40,1. hh (-): ( in a complex) hh (+): 2. Smo activated: coupled with a complex 3. hh (-): (1) Ci N ter. transcription repressor (Ci 75) (2) Ci in cytoplasm ( Cos2 / Fu / Su / Ci complex anchored on
40、microtubule) 4. hh (+): Costa / Fu / Su / Ci dissociated from microtubule Ci (Full length): enters nucleus Transcription of genes ( ptc, Hip and growth genes),Activation of hh Pathway,hh Ptc Smo*,hh - Ptc,Smo*,Smo*,Fu: Fused, S / T PK Su: Suppressor of Fu Costa 2 ( Cos. 2 ) : Kinase like Ci: Cubitus interuptus, ZF TA Gli in mammal,PKA,41,Su,Fused (Fu),Costa 2,Smoothened (Smo),Patched (Prc),Hedgehog (Hh),C i,C i,Growth gene product,Ptc Hip,Patehed: Disrfunctin in BCC Smo: mutated in breast Hedgehog Pathway,MiT microtubule,Mit,42,