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POST-TRANSLATIONAL PROCESS YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE

POST-TRANSLATIONAL PROCESS YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE THE DEPARTMENT OF MEDICAL BIOLOGY ASST. PROF. DR. ENDER ŞİMŞEK. Protein: Post-translational Modification. Gene encoding region (ORF) ↓ transcription mRNA

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POST-TRANSLATIONAL PROCESS YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE

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  1. POST-TRANSLATIONAL PROCESS YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE THE DEPARTMENT OF MEDICAL BIOLOGY ASST. PROF. DR. ENDER ŞİMŞEK

  2. Protein: Post-translational Modification Gene encoding region (ORF) ↓transcription mRNA ↓translation Protein (nascentprotein, precursorprotein) ↓proteinprocessing, post-translational modification Mature protein ↓folding Biologically active protein

  3. Post-translational Modification • 1. Numerous and diverse • 2. Change the charge, conformation or size of protein molecule

  4. Effects of Post-translational Modification • 1. Stability of protein • 2. Biochemical activity (activity regulation) • 3. Protein targeting (protein localization) • 4. Proteinsignaling (protein-proteininteraction, cascade amplification)

  5. Types of Post-translational Modification

  6. Modification Involving Peptide Bonds Cleavage (limited proteolysis) • Peptide Bonds Cleavage (limited proteolysis, specific and • well-regulated ) • Peptide Bond Isomerization (Intramolecular) 3. Peptide Bond Formation, Transpeptidation

  7. Peptide Bonds Cleavage (limited proteolysis, specific and • well-regulated ) • Signal leader peptide removed by signal peptidase (both in prokaryotes and eukaryotes) Precursor protein → mature protein (Insulin) • Zymogen → active enzyme • Trypsinogen → Trypsin • Pepsinogen → Pepsin • Prohormone → Hormone • Polyprotein → neuropeptides (peptide hormone ) conversion

  8. * First, preproinsulin as a form of straight chain is synthesized on the ribosome. * Then, they are converted to proinsulin in the lumen of ER. They are stored as vesicles in the Golgi. * Finally, when they are needed in the body, by hydrolysis the 'pro' portion or C peptide fragment is separated. They are sent out of the cell by exocytosis. * The active form is insulin destroyed by insulinase.

  9. Modifications Involving Amino and Carboxyl Termini • The N terminus • The C Terminus

  10. N-Formyl- (C1) N-Acetyl- (C2) N-Acyl- (C2, C4, C6, C8, C10) N-Lauroyl- (C12) N-Myristoyl- (C14) N-Tetradeca (mono and di)enoyl- (C14：1；C14：2) N-Aminoacyl- N-α-Ketoacyl- N-Methyl- N-Pyrrolidone carboxyl- N-Glucuronyl- N-Glycosyl- Modifications Involving Amino and Carboxyl Termini • The N terminus：

  11. Amide • O-(ADP-ribosyl)- • O-Methyl- • -(N-Ethanolamine-glycan-phosphoinositides) • -(Nα-TyT) • The C Terminus：

  12. C. Modifications Involving Individual Amino Acid (Side Chains) • Arginine • Histidine • Lysine • Asparagine 9. Methionine 3. Aspartate • Phenylalanine 4. Cysteine 11. Proline • Glutamate • Serine • Glutamine

  13. Nω-(ADP-ribosyl)- Nω-Methyl- Nω-Dimethyl- Nω-Nω’-Dimethyl- Ornithine Citrulline Nω-Phosphoryl- • Arginine：

  14. N-Glycosyl- Aspartate N-Methyl- Nε-(β-Aspartyl)lysine erythro-β-Hydroxy- N-(ADP-ribosyl)- • Asparagine：

  15. D-Asp (racemization) β-Carboxy- erythro-β-Hydroxy- β-Methylthio- O-Phosphoryl- O-Methyl- 3. Aspartate：

  16. Cystine S-γ-Glutamyl- S-(2-Histidyl)- S-(3-Tyr) S-(sn-1-Glyceryl)- S-(sn-1-Diacylglyceryl)- S-(sn-1-{2,3,-Di-O-[3’ ,7’ ,11’ .15’- tetramethylhexadecyl]}glyceryl)- S-Palmitoyl- S-Farnesyl- S-Geranylgeranyl- S-Heme S-Phycocyanobilin S-p-Coumaroyl S-(6-Flavin [FMN]) S-(8α-Flavin [FAD]) S-Coenzyme A S-(ADP-ribosyl)- S-Glycosyl- Dehydroalanine Lysinoalanine Lanthionine Selenocysteine 4. Cysteine：

  17. Glutamate： • O-(ADP-ribosyl) • γ-Carboxy- • O-Methyl- • Nα-(γ-Glutamyl)-Glu1-5 • Nα-(γ-Glutamyl)-Glu3-34 • N- (γ-Glutamyl)ethanolaminephosphate) • S-γ-Glutamyl-Cys is listed under Cys

  18. Glutamine： • Glutamate • Nε-(γ-Glutamyl)lysine • N-(γ-Glutamyl)-L-ornithine • N-(γ-Glutamyl)polyamine • N,N-(Bis-γ-glutamyl)polyamine • N5-Methyl-

  19. Histidine： • Diphthamide • Nτ-(ADP-ribosyl)diphthamide • N-Phosphoryl- • Nπ-Methyl- • 4-Iodo-and diiodo- • Nτ- and Nπ –(8α-flavin [FAD]) • Nπ-(8α-Flavin[FMN])

  20. Nε-Acetyl- Nε-( Nα-Monomethylalanyl)- Nε-Murein (peptidoglycan) Nε-Lipoyl- Nε-Biotinyl- Nε-Ubiquitinyl- Nε-Phosphoryl- Nε-Phosphopyridoxyl- Nε-Retinyl- Nε-Glycosyl- Nε-Mono-, di- , trimethyl- Hypusine：Nε-(4-amino-2-hydroxybutyl)- Allysine δ-Hydroxy- δ-Hydroxyallysine Cross-links (desmosines, syndesines, pyridinolines) δ-Glyxosyloxy- • Lysine：

  21. Sulfoxide 9. Methionine： • Phenylalanine： • β-Glycosyloxy-

  22. 11. Proline： • 3-Hydroxy- • 4-Hydroxy- • 3,4-Dihydroxy- • O4-Arabinosylhydroxy- • O4-Galactosylhydroxy- • O4-Glucosylhydroxy-

  23. Selenocysteine O-Phosphoryl- O-Pantetheinephosphoryl- O-(GlcNAc-1-phosphoryl)- O-(Glycerol-1-phosphoryl)- O-Methyl- O-Glycosyl- Alanino(τ- or π-histidine) Lanthionine O-Acetyl- O-Fattyacyl- • Serine：

  24. EXAMPLE 1: Phosphorylation

  25. EXAMPLE 2: Carbonylation

  26. EXAMPLE 3: Methylation

  27. How are modifications made ?

  28. A. Nonenzymatic Reaction How are modifications made ? B. Enzymatic Reaction 1. Irreversible, Unidirectional Reaction (Permanently Modified) 2. Irreversible, Bi-directionalReaction. (Signal Amplification) 3. Reversible Reaction

  29. A. Nonenzymatic Reaction • deamidation：Asn, Gln • racemization：Asp, Ser • dehydroalanine：Cys, phosphor-Ser • slow oxidation：Cys, His, Met • slow cleavage and permutation of peptide bonds • reducing sugar reaction with NH2-group of aa’s or side chains (Lys)：Maillard reaction (Browing reaction)；Schiffs base reaction.

  30. B. Enzymatic Reaction • N-linked glycosylation • Carboxyl methylation • S-isoprenylation-Cys 1. Irreversible, Unidirectional Reaction (Permanently Modified)

  31. Protein GlycosylationCommon in Eukaryotic Proteins

  32. N-Linked Glycans • N-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

  33. 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-galactosaminyltransferases, then further elaborated by a series of glycosyltransferasesto 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 protein's primary sequence

  34. P P P P P ProteinlerinGlikolizasyonu N-linked oligosakkaridlerinBiyosentezi(ilk 7 basamak) Dolikol fosfat (polyprenol lipid carrier) (2) UDP- ER lumen (1) UMP, (1) UDP (5) GDP- (5) GDP reorientation Sitosol N-asetilglukozamin (GlcNAc) = Mannose = Monosakkaridlerdennükleotidşekerler spesifikglycosyltransferasesaracılığıile eklenir

  35. PP P P PP P P N-linked oligosakkaridlerin Biyosentezi (ikinci 7 basamak) ER lumen Dolicol fosfatlar ER lümenindeki şeker vericileridir; lümende transloke olmadan sitozolde sentezlenirler (4) Dolicol-P-mannoz = Dolicol-P-glukoz = (3) PP Sitosol

  36. Oligosakkaritlerin proteinlere transferi PP Büyüyen polipeptide oligosakkarit transferi ER lumen Asn I X I Ser (Thr) Bağlantı asparajinin amid grubuna olur. Prolin olmamak kaydıyla herhangi bir aa takip eder ve sonraki aa serin veya threonin aminoasidi olmalıdır. Sentezi takiben, protein Golgi kompleksine transfer olur ve burada budanır ve yeni oligosakkarid kompleksleri eklenir. Sitosol

  37. Oligosakkarit kompleks oluşumu Asn I X I Ser (Thr) Glkozidazlar tarafından budanma; glikoziltransferaz tarafından eklenme = yaygınkor yapısı Asn I X I Ser (Thr) Golgi lumen Oligosakkarit kompleksi fruktoz = galaktoz = sialik asid = Golgi membranını geçerken nükleotit şekerlei transloke olur. Sitosol Karbonhidrat tipi proteinin membranamı, vezikülemi yada direkt olarak sekrete edilip edilmeyeceğini belirler

  38. P P Proteinlerin lizozomlara yönlendirilmesi • mannose-6-phosphate içeren • proteinler lizozomlara yönlendirilir. • Fosfat grupları mannoza eklenir (UDP N-asetil glukozaminden alınan fosfat- Nasetil glukozamin yapısı) • Nasetilglukozaminler uzaklaştırılır. Asn UDP- Asn P Asn P

  39. The Topology of HBV Surface Antigens n-4 (n-146) (Lambert and Prange, 2001)

  40. Consensus sequence: Asn - X - Ser/Thr N-Linked Glycosylation Biological importance: - Protein folding - Protein stability - Sorting signal Experimental tool: - Reporter for a protein’s progress through the secretory pathway

  41. Moleküler şaperonlar, proteinlerin sentezinde, taşınmasında, polimerlerinin oluşmasında ve denatüre proteinlerin yeniden doğal şekillerine dönüşmesinde (renatürasyonda) rol oynamaktadırlar.

  42. N-Linked Glycosylation Pathway (Mehta et al., 1998) DRUGS: UN NB DN DM HBV rcDNA (3.5kb)

  43. Proper Glycoprotein Folding through Chaperone Interactions (Helenius and Aebi, 2004)

  44. Phosphorylation (protein kinase) / Dephosphorylation (phosphatase)：Ser, Tyr, Thr. • Uridylyl and adenylyl transfer in bacterial glutamine synthetase 2. Irreversible, Bi-directionalReaction. (Signal Amplificaion)

  45. Protein Phosphorylation Phosphorylation is a highly effective means of regulating the activity of target proteins. ~ 30% eukaryotic proteins are phosphorylated. Protein kinases: The enzymes that catalyze phosphorylation reactions. one of the largest protein families ~ 100 in yeast ~ 500 in human beings Kinases: specificity Ser/Thr kinases: acceptor is -OH of Ser or Thr Tyr kinases: acceptor is -OH of Tyr Tyr kinases are unique to multicellular organisms, important in growth regulation. Tyr kinase mutations often occur in cancer cells

  46. Protein Phosphorylation Phosphorylation Dephosphorylation

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