分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi

分子遗传学课件Protein translation for mol med genetics final part2分子遗传学课件Protein translation for1分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi2PART I:Protein Synthesis ComponentsPART II:Protein Synthesis ProcessPART III:Protein Synthesis RegulationPART IV:Posttranslational Processing and TargetingPART V:Protein Synthesis in MedicinePART I:Protein Synthesis Comp3PART IV:Posttranslational Processing and Targeting分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi4From DNA to proteinFrom DNA to protein56Fate of nascent polypeptidesAssociation of nascent polypeptide with various chaperone systems commits them to folding pathwaysIf proteins fail to fold(one third of the nascent polypeptides),they are recognized and targeted for degradation6Fate of nascent polypeptidesAThe macromolecules assisting the formation of protein secondary structure includeMolecular ChaperonProtein Disulfide Isomerase(PDI)Peptide Prolyl Cis-trans Isomerase(PPI)4.1 Protein Folding Newly synthesized polypeptides must form higher order structure before they become functional proteinsThe macromolecules assisting t78Proteins are assisted in folding by molecular chaperonesHeat shock proteins,Hsp60,Hsp70 and Hsp90 are three main classes Hsp70 recognizes exposed,unfolded regions of new protein chains-especially hydrophobic regions It binds to these regions,protecting them until productive folding reactions can occur 4.2.1 Chaperons8Proteins are assisted in foldMechanismhollow cylinderHsp60Mechanismhollow cylinder9GroEL forms two stacked 7-membered rings of 60 kD subunits;GroES is a dome on the topThe GroES-GroEL Complex(Hsp60)GroEL forms two stacked 7-memb1011Model of the GroEL cylinder(blue)in action Nascent protein apparently binds reversibly many times to the walls of the donut structure,each time driven by ATP hydrolysis,eventually adopting its folded structure,then being released from the GroES-GroEL complex 11Model of the GroEL cylinder 12Eucaryotic Hsp90 ChaperonesHsp90s account for 1-2%of total cytosolic proteinSignal transduction molecules such as tyrosine kinase receptors,steroid hormone receptors,non-receptor tyrosine kinases are clients for Hsp9012Eucaryotic Hsp90 ChaperonesH分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi134.2.2 Protein disulfite isomerases pattern disulfide bondsCellular environment is reducingCysteines are oxidized to form S-S bondsIn eukaryotes,protein disulfide isomerase,PDI,used for oxidation in the endoplasmic reticulum(ER)4.2.2 Protein disulfite isomer14The corrective disulfide is very important for the structure of secreted proteins and membrane proteins.The corrective disulfide is ve154.2.3 Peptidyl prolyl isomerization16Isomerization of prolinesMost peptide bonds are trans(100 x more stable than cis).When second residue is proline,trans form is only 4x more stableIn native proteins,cis-proline peptides are stabilized by tertiary structure but in unfolded state there is an equilibrium between cis and trans isomersCis-trans isomerization of proline peptides,catalyzed by PPI,is the rate-limiting step in folding for some proteins4.2.3 Peptidyl prolyl isomerizCyclosporine A and Cyclophilin FK506 and FKBP12Cyclosporine A and Cyclophilin174.3 Posttranslational Processing and Modifications of Newly Synthesized PolypeptidesN-and C-terminus processing and/or modification Trimming of peptides through proteolytic cleavageCovalent modification of some amino acids(phosphorylation,methylation,acetylation,glycosylation)4.3 Posttranslational Proces18 N-and C-terminus processing and/or modificationl Removing N-fMet,(in prokaryotes)or N-Met(in eukaryotes),or several amino acid residues at the N-terminal(in both prokaryotes and eukaryotes)l Acetylation of N-terminal amino acid residue in more than 50%eukaryotic proteinl C-terminal processing may also occurs N-and C-ter19Methionine aminopeptidase 2FumagillinAnti-angiogenesis acitivityMethionine aminopeptidase 2Fum20POMC,a polypeptide hormone precursor,is cleaved into different peptide hormones in different tissues.NCSignal peptideKRKR103peptideACTH-LT-MSH-MSHEndophinPolypeptides may undergo proteolytic processing to produce several peptidesPOMC,a polypeptide hormone pr21Posttranslational ModificationWhat is it?Addition of groups or deletion of parts to make a finished proteinWhat groups?How much?Where?-methyl -acetyl -glyco -phosphoAnd many moreWhat purpose?-targeting(eg.some lipoproteins)-stability(eg.secreted glycoproteins)-function(eg.surface glycoproteins)-control of activity(eg.clotting factors,caspases)Posttranslational Modification22Histone ModificationHistone Modification23Histone Code Hypothesis Histones can be modified by post-translational modifications (PTMs),including acetylation,methylation,phosphorylation and ubiquitination(mainly in N-terminal)The histone code hypothesis:specific PTMs regulate gene expression by two mechanisms:(1)changing the chromatin structure into activated or repressed transcriptional state (2)acting as a docking site for transcriptional regulatorsH3K4meH3K36meH3K9meH3K27meEuchromatinEuchromatinHeterochromatinHeterochromatinLow acetylationH3K9me,H3K27me,H4K20meHigh acetylationH3K4me,H3K36me,H3K79meHistone Code Hypothesis Histon24PhosphorylationP-SITE:S/T/YPhosphorylationP-SITE:25Amplification or over-expression of HER2 in 30%breast cancerTherapy:monoclonal antibody trastuzumab(Herceptin)Pertuzumab,which inhibits dimerization of HER2 and HER3 receptorstamoxifenAmplification or over-expressi26Protein Glycosylation Common in Eukaryotic Proteins27NitrogenAsparagineSerine and ThreonineProtein Glycosylation Common分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi28N-Linked GlycansN-linked glycans are covalently attached to Asn residues within a consensus sequence(Asn-Xaa-Ser/Thr),enabling prediction of the modification sites by protein sequence analysis All N-linked glycans share a common pentasaccharide core(GlcNAc2Man3)recognized by lectins and N-glycanase enzymes(PNGase F)These reagents have been used to visualize proteins bearing N-linked glycans from cell or tissue lysates and to enrich them for mass spectrometry analysis N-Linked GlycansN-linked glyca29O-Linked GlycansComparable tools are lacking for the study of proteins bearing O-linked glycans.Mucin-type,the most prevalent O-linked glycosylation is characterized by an N-acetylgalactosamine(GalNAc)residue-linked to the hydroxyl group of Ser or Thr.GalNAc residue is installed by a family of 24 N-acetyl-galactosaminyl transferases,then further elaborated by a series of glycosyl transferases to generate higher-order O-linked structures.Because of the complex biosynthetic origin,O-linked glycans are not installed at a defined consensus motif and their presence cannot be accurately predicted based on the proteins primary sequence O-Linked GlycansComparable too30The correctly folded proteins need to be transported to special cellular compartments to exert desired biological functions.AAs sequence on the N-terminus that directs proteins to be transported to proper cellular target sites is called signal sequence.4.4 Protein Targeting The correctly folded proteins 31 Signal sequencesAll the secretory proteins have the signal sequences.Consist of 13-36 AA in three regions Positively charged AA at N-terminusHydrophobic core of 10-15 AA in the medial regionSmall polar AA at C-terminus Signal sequencesAll the secre32 Signal sequence for ERCleavage site Signal sequence for ERCleavag33 a.Secreted protein into ERSignal recognition particle(SRP)a.Secreted protein into ERSi34分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi35分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi36ER membraneER membrane37分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi38分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi39Ribosome dissociatesRibosome dissociates40分子遗传学ppt课件Protein-translation-for-mol-med-genetics-fi41 Secretory proteins are transported to the cell periphery Secretory proteins are transp42 b.Mitochondrial proteinMitochondrial proteins in cytosol are present in precursor forms.Signal sequence of 20-25 AA at N-terminus are rich in Ser,Thr,and basic AA.b.Mitochondrial proteinMitoc43 b.Mitochondrial protein b.Mitochondrial protein44 c.Nuclear proteinK-K/R-X-K/R Nuclear Localization Sequence c.Nuclear proteinK-K/R-X-K/R45D.Nuclear Export SignalLXXXLXXLXLD.Nuclear Export SignalLXXXLX46PART V:Protein Synthesis in MedicinePART V:47 1.Molecular diseases are associated with defective proteinsMutations on gene coding regionsSilent mutations:code for the same amino acid.Missense mutations:code for a different amino acid.Nonsense mutations:introduce a stop codon and can truncate the protein.Abnormal protein foldingCystic fibrosis caused by deletion of Phe508 in CFTR,resulting in improper protein folding 1.Molecular diseases are ass48Online Mendelian Inheritance in Man(OMIM)is a database that catalogues all the known diseases with a genetic componentOnline Mendelian Inheritance i49Uniprot(http:/www.uniprot.org)Uniprot(http:/www.uniprot.or50tRNA structure and modifications tRNA structure and modificatio51Human disease associated with tRNA modificationHuman disease associated with 52Known chemical structure of tRNA modification linked to human diseaseKnown chemical structure of tR53Ribosomopathies At least 50%of patients with Diamond-Blackfan anemia(先天性再生障碍性贫血)carry mutations in ribosomal proteins Treacher-Collins syndrome,North American Indian childhood cirrhosis,chromosome 5q-syndrome isolated congenital asplenia Ribosomopathies At least 50%o54Congenital AspleniaIn isolated congenital asplenia,the most recently discovered ribosomopathy,haploinsufficiency of the ribosomalprotein RPSA prevents splenic development.These patients are prone to severe bacterial infections because they lack a spleen,but they are otherwise healthyand have no other observable developmentalanomalies Congenital AspleniaIn isolated55Diamond-Blackfan anemia(戴戴-布二氏贫布二氏贫血血)In the case of Diamond-Blackfan anemia,mutations in any of 11 different ribosomal proteins lead to bone marrow failure.Diamond-Blackfan anemia(戴-布二氏56CirrhosisIn North American Indian childhood cirrhosis,a mutation in the ribosome biogenesis factor hUTP4/Cirhin causes biliary cirrhosis,for which the only treatment is liver transplantation required by early adolescence.Little is known about the molecular mecha-nisms that lead to this disease.CirrhosisIn North American Ind57Shwachman-Bodian-Diamond syndrome(许氏症氏症)Shwachman-Bodian-Diamond syndrome arises from mutations in the SBDS protein,which is involved in large ribosomal subunit maturation.Patients with this disorder suffer not only from dysfunction of the pancreas,but also from bone marrow failure,skeletal abnormalities,and an enlarged liver.Shwachman-Bodian-Diamond syndr58Treacher-Collins syndrome(颌面部骨发育颌面部骨发育不全综合不全综合征征)Treacher-Collins syndrome often results from haploinsufficiency in TCOF1,the gene that encodes Treacle,a nucleolar protein involved in pre-rRNA synthesis.Treacher-Collins syndrome(颌面部592.Many viruses use the host cell protein synthesis machinery(1)Virus mRNA is more efficiently translated than host cell mRNA.(2)Viruses make abundant mRNA.(3)Some viruses can inhibit host cell mRNA binding to 40S subunit.2.Many viruses use the host c60 3.Protein Synthesis can be inhibited by antibiotics and toxinsThe protein synthesis is highly regulated.This process can also be the primary target for many toxins,antibiotics and interferons.These interferons interact specifically with proteins and RNAs to interrupt the protein synthesis.3.Protein Synthesis can be i61 nametargetfunctionTetracycline四环素30Sblock the A site to prevent binding of AA-tRNA with 30SStreptomycin链霉素30SInhibits initiationChloromycetin氯霉素50Sblock the peptidyl transferase,and inhibit the elongationCycloheximide放线菌酮60S(Eu)block the peptidyl transferase,and inhibit the elongationPuromycin嘌呤霉素50 S(Pro)and 60S(Eu)release the prematured peptide,and terminates elongationErythromycin红霉素50 SInhibit the translocaseAntibiotics nametargetfunctionTetracyclin62Antibiotics bound to the decoding center of the eubacterial 70S ribosomeVIO:viomycin;PAR:paromomycin;TER:thermorubinAntibiotics bound to the decod63Antibiotics bound in the exit tunnel and the peptidyl transferase center of eubacterial 70S ribosome.erythromycin(ERY,orange),clindamycin(CLI,pink),and chloramphenicol(CAM,purple)Antibiotics bound in the exit 64Antibiotics that compete with the binding of the deacylated tRNA to the E-site13-deoxytedanolide(DTL)cycloheximide(CXM)Antibiotics that compete with 65 It has a similar structure to Tyr-tRNA.It works for both prokaryotes and eukaryotes.Puromycin It has a similar structure to66Some toxins,such as plant protein Ricin(篦麻毒素),is among the most toxic substance known,which acts on 60s subunits.ToxinsRicinus Communiscastor-oil plantSome toxins,such as plant pro67rRNA N-glycosylase activityThe ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes of 60srRNA N-glycosylase activity68Diphtheria toxinDiphtheria toxin is an exotoxin secreted by Corynebacterium diphtheriae,the pathogen bacterium that causes diphtheria.Unusually,the toxin gene is encoded by a bacteriophage(a virus that infects bacteria).The toxin causes the disease diphtheria in humans by gaining entry into the cell cytoplasm and inhibiting protein synthesisDiphtheria toxinDiphtheria tox69Interferons are cytokines produced during immune response to antigens,especially to viral infections.InterferonInterferons are cytokines prod70InterferonInterferon71mRNAmRNA72Pateamine A(PatA)Northcote,P.T.et al.(1991)Tetrahedron Lett.Romo et al.(1998)JACSoCell proliferation-IC50=0.15 ng/mL in murine cell line(P388).oImmunosuppressive-IC50=2.6 nM in murine MLR and blocked oMouse skin graft rejection better than CsAoInhibition of TCR-mediated IL-2 productionoSelective Inhibition of NF-kB activationoPotent inhibition of tumor cell proliferationPateamine A(PatA)Northcote,P73Mol Cancer Ther.2009 May;8(5):1250-60PatA Efficiently Inhibits Melanoma In VivoMol Cancer Ther.2009 May;8(5)74Malina A et.al(2011)Oncotarget Translation Initiation is A Novel Target for TherapyMalina A et.al(2011)Oncotarg75Summary of TranslationComponents needed in translationmRNA,AA,tRNA,Ribosome,Enzymes and Factors,Energy melucuesTranslation processesInitiation,elongation and terminationTranslation regulationInitiation,elongation and terminationPosttranslational ProcessingFolding,Cleavage,modificationProtein targetingER,Golgi,membrane,mitochondria,nucleus Translation and MedicineSummary of TranslationComponen76