1、 PART I: Protein Synthesis Components PART II: Protein Synthesis Process PART III: Protein Synthesis Regulation PART IV: Posttranslational Processing and Targeting PART V: Protein Synthesis in MedicinePART IV: Posttranslational Processing and TargetingFrom DNA to protein4Fate of nascent polypeptides
2、 Association of nascent polypeptide with various chaperone systems commits them to folding pathways If proteins fail to fold (one third of the nascent polypeptides), they are recognized and targeted for degradation The macromolecules assisting the formation of protein secondary structure include Mol
3、ecular Chaperon Protein Disulfide Isomerase(PDI) Peptide ProlylCis-trans Isomerase(PPI)4.1Protein Folding Newly synthesized polypeptides must form higher order structure before they become functional proteins6 Proteins are assisted in folding by molecular chaperones Heat shock proteins, Hsp60, Hsp70
4、 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 occur4.2.1 ChaperonsMechanismhollow cylinderHsp60GroEL forms two stacked 7-membered ri
5、ngs of 60 kD subunits; GroES is a dome on the topThe GroES-GroEL Complex (Hsp60)9Model 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
6、 being released from the GroES-GroEL complex 10Eucaryotic Hsp90 Chaperones Hsp90s account for 1-2% of total cytosolicprotein Signal transduction molecules such as tyrosine kinase receptors, steroid hormone receptors, non-receptor tyrosine kinasesare clients for Hsp904.2.2 Protein disulfiteisomerases
7、 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)The corrective disulfide is very important for the structure of secreted proteins and membrane proteins.
8、4.2.3 Peptidylprolyl isomerizationIsomerization of prolines Most peptide bonds are trans (100 x more stable than cis).When second residue is proline, trans form is only 4x more stable In native proteins, cis-proline peptides are stabilized by tertiary structure but in unfolded state there is an equi
9、librium between cis and trans isomers Cis-transisomerization of proline peptides, catalyzed by PPI, is the rate-limiting step in folding for some proteinsCyclosporine A and CyclophilinFK506 and FKBP124.3 Posttranslational Processing and Modifications of Newly Synthesized Polypeptides N- and C-termin
10、us processing and/or modification Trimming of peptides through proteolytic cleavage Covalent modification of some amino acids (phosphorylation, methylation, acetylation, glycosylation)N- and C-terminus processing and/or modificationlRemoving N-fMet, ( in prokaryotes ) or N-Met ( in eukaryotes ), or
11、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 occursMethionine aminopeptidase 2FumagillinAnti-angiogenesis acitivityPOMC, a polypeptide hormone pre
12、cursor, is cleaved into different peptide hormones in different tissues.NCSignal peptideKRKR103peptideACTH-LT-MSH-MSHEndophinPolypeptides may undergo proteolytic processing to produce several peptidesPosttranslational Modification What is it ?Addition of groups or deletion of parts to make a finishe
13、d protein What groups ? How much ? Where ? - methyl-acetyl-glyco - phosphoAnd many more What purpose ? -targeting (eg. some lipoproteins) -stability (eg. secreted glycoproteins ) - function (eg. surface glycoproteins) - control of activity (eg. clotting factors, caspases)Histone ModificationHistone
14、Code HypothesisHistones 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 structur
15、e into activated or repressed transcriptional state (2) acting as a docking site for transcriptional regulatorsH3K4meH3K36meH3K9meH3K27meEuchromatinEuchromatinHeterochromatinHeterochromatinLow acetylationH3K9me, H3K27me, H4K20meHigh acetylationH3K4me, H3K36me, H3K79mePhosphorylationP-SITE:S/T/YAmpli
16、fication or over-expression of HER2 in 30% breast cancerTherapy:monoclonal antibody trastuzumab (Herceptin)Pertuzumab, which inhibits dimerization of HER2 and HER3 receptorstamoxifenProtein GlycosylationCommon in Eukaryotic Proteins25NitrogenAsparagineSerine and ThreonineN-Linked Glycans N-linked gl
17、ycans 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 pentasaccharidecore (GlcNAc2Man3) recognized by lectins and N-glycanaseenzymes (PNGase F) These
18、reagents have been used to visualize proteins bearing N-linked glycans from cell or tissue lysates and to enrich them for mass spectrometry analysis O-Linked Glycans Comparable tools are lacking for the study of proteins bearing O-linked glycans. Mucin-type, the most prevalent O-linked glycosylation
19、 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-galactosaminyltransferases, then further elaborated by a series of glycosyltransferasesto generate higher-order O-linked structures. Beca
20、use 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 The correctly folded proteins need to be transported to special cellular compartments to exert desired biol
21、ogical 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 Signal sequences All the secretory proteins have the signal sequences. Consist of 13-36 AA in three regions Positively charged AA a
22、t N-terminusHydrophobic core of 10-15 AA in the medial regionSmall polar AA at C-terminusN-terminushydrophobiccoreSignal sequence for ERCleavage sitea. Secreted protein into ERSignal recognition particle (SRP)mRNAencoded signal sequencesignal sequence at end of protein emerging from ribosomeSignal r
23、ecognition particle (SRP)signal sequence at end of protein emerging from ribosomeBinding site for SRP receptorSignal peptidaseSRP receptorGTPER membraneSignal peptidaseGDP + PiSignal peptidaseSignal peptidase cleaves signal peptideRibosome dissociatesCompleted polypeptide chainSecretory proteins are
24、 transported to the cell peripheryb. Mitochondrial protein Mitochondrial 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 proteinc. Nuclear proteinK-K/R-X-K/R Nuclear Localization SequenceD. Nuclear Exp
25、ort SignalLXXXLXXLXLPART V: Protein Synthesis in Medicine1. Molecular diseases are associated with defective proteins Mutations on gene coding regions Silent mutations: code for the same amino acid. Missense mutations: code for a different amino acid. Nonsense mutations: introduce a stop codon and c
26、an truncate the protein. Abnormal protein folding Cystic fibrosis caused by deletion of Phe508 in CFTR, resulting in improper protein foldingOnline Mendelian Inheritance in Man (OMIM) is a database that catalogues all the known diseases with a genetic componentUniprot(http:/www.uniprot.org)Ribosomop
27、athies At least 50% of patients with Diamond-Blackfananemia(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 aspleniaCongenital AspleniaIn isolated congenital asplenia, the most recently
28、 discovered ribosomopathy, haploinsufficiencyof the ribosomalprotein RPSA prevents splenic development.These patients areprone to severe bacterial infections because they lack a spleen,but they are otherwise healthyand have no other observable developmentalanomalies Diamond-Blackfananemia(戴戴-布二氏贫布二氏
29、贫血血)In the case of Diamond-Blackfananemia, mutations in any of 11 different ribosomal proteins lead to bone marrow failure. Cirrhosis In North American Indian childhood cirrhosis, a mutation in the ribosome biogenesis factor hUTP4/Cirhincauses biliary cirrhosis, for which the only treatment is liver
30、 transplantation required by early adolescence. Little is known about the molecular mecha- nisms that lead to this disease. Shwachman-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. Pa
31、tients with this disorder suffer not only from dysfunction of the pancreas, but also from bone marrow failure, skeletal abnormalities, and an enlarged liver.Treacher-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 encode
32、s Treacle, a nucleolar protein involved in pre-rRNA synthesis. 2. 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.3. Protein S
33、ynthesis can be inhibited by antibiotics and toxins The protein synthesis is highly regulated. This process can also be the primary target for many toxins, antibiotics and interferons. The interferons interact specifically with proteins and RNAs to interrupt the protein synthesis. 53PAsitesitechloro
34、m ycetins tre p to m y c in a n d k a ra m y c inPuromycinT e t r a c y c lin ecycloheximideAntibioticsnametargetfunctionTetracycline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 peptidyltransferase,and inhibit the elon
35、gationCycloheximide放线菌酮60S (Eu)block the peptidyltransferase, 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 It has a similar structure to Tyr-tRNA. It works for both prokary
36、otes and eukaryotes.Puromycin Some toxins, such as plant protein Ricin (篦麻毒素), is among the most toxic substance known, which acts on 60s subunits. ToxinsRicinusCommuniscastor-oil plantrRNA N-glycosylase activityThe ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotid
37、es universally found in eukaryotic ribosomes of 60sDiphtheria toxinEF-2active+Diphtheria toxinOOHOHCH2OADPNCNH2OOOHOHCH2OADPEF-2(inactive)NCNH2O+Diphtheria toxin is an exotoxin secreted by Corynebacteriumdiphtheriae, the pathogen bacterium that causes diphtheria. Unusually, the toxin gene is encoded
38、 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 synthesis Interferons are cytokines produced during immune response to antigens, especially to viral infections.InterferonInterferonpr
39、otein kinaseeIF2eIF2-PATPADPactiveinactiveInterferoninteferon2,5-AsynthetaseApppATPAppppp25525AA2,5-ARNaseLRNaseLinactiveactivedegrademRNAPateamineA (PatA)SNOOMeOONMe2MeH2NMeMeMePateamine ANorthcote, P.T. et al. (1991) Tetrahedron Lett.Romoet al. (1998) JACSoCell proliferation-IC50 = 0.15 ng/mL in m
40、urine 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-kBactivationoPotent inhibition of tumor cell proliferationMol Cancer Ther. 2009 May;8(5):1250-60PatA Ef
41、ficiently Inhibits Melanoma In VivoMalina A et.al (2011) OncotargetTranslation Initiation is A Novel Target for TherapySummary of Translation Components needed in translation mRNA, AA, tRNA, Ribosome, Enzymes and Factors, Energy melucues Translation processes Initiation, elongation and termination Translation regulation Initiation, elongation and termination Posttranslational Processing Folding, Cleavage, modification Protein targeting ER, Golgi, membrane, mitochondria, nucleus Translation and Medicine
