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Protein_ Post-translational Modification.ppt

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					Protein: Post-translational Modification

             林富邦老師
     Gene encoding region (ORF)
                   ↓transcription
                 mRNA
                   ↓translation
Protein (nascent protein, precursor protein)
                     ↓protein processing, post-translational modification
             Mature protein
                   ↓folding
        Biological active protein
Post-translational Modification
n   1. numerous and diverse

n   2. change the charge, conformation or size of
       protein molecule
Effects of Post-translational Modification

n   1. stability of protein

n   2. biochemical activity (activity regulation)

n   3. protein targeting (protein localization)

n   4. protein signaling (protein-protein
      interaction,cascade amplification)
Why protein post-translational modifications
are made? (Biological functions)
     A. Regulation (interconvertable modifications)

n    Monocyclic cascade (allosteric effectors)
    -P/de-P receptor-associated Tyr-Kinase coupled cascade

n   Cyclic cascade
    ADP-ribosylation / poly-ADP-ribosylation
    Coordinated Glycogen phosphorylase / Glycogen synthase

n   Unidirectional cascade
    Proteolytic activation
     B. Cross-links
Stabilize or fix certain folded str. (Cofactors covalent binding)
     n   Disulfide bond-cross links (Cys-Cys)

     n   Isopeptide (N-(γ-Gln)-Lys

     n   Transamidation (Gln→Lys or Ornithine-α-NH2)
         (Transglutaminases)
          Ø   Blood clotting factor VIII – coagulation
          Ø   Tissue Transglutaminase reaction
          Ø   Cell proliferation, aging, endocytosis, secretion, differentiation,
              apotosis, programmed cell death
C.      Covalent Cofactors
n    Biotinyl lysine (Carboxylase, transcarboxylases)

n    Cys-bound linear tetrapyrrole (phycobiliproteins light-
     harvesting system of photosynthetic microorganisms)

n    FAD-linked His, Cys, or Tyr (DHase, Oxidase)

n    FMN-linked Cys

n    Heme-covalent bound (Cyt. C)
D.      Membrane Anchors
n   α-NH2 myristoyl

n   Cys- fattyacyl thioether

n   Ser- palmitate or other fatty acids esters
    Thr-

n   C-terminal glycophospholipids

n   C-terminal Cys – prenylated group
    Farnesyl C15, Geranylgeranyl C20
E. Signaling, Recognition and
    Structural Amplification
F.     Protein Turnover (Protein
       Degradation)
n   Spontaneous Oxidation: Cys, His, Tyr, Met

n   Ubiquitination: Lys
G. Others
n   Iodination: Tyr, (Thyroid hormones)

n   Sulfation or methylation; Tyr / secreted proteins
Types of Post-translational
      Modification
  A. Modification Involving Peptide
     Bonds Cleavage (limited proteolysis)
• Peptide Bonds Cleavage (limited proteolysis, specific and
   well-regulated )
  n   Signal leader peptide removed by signal peptidase (both in
      prokaryotes and eukaryotes)
      Precursor protein → mature protein (Insulin)
  n   Zymogen → active enzyme
  n   Trypsinogen → Trypsin
  n   Pepsinogen → Pepsin
  n   Prohormone → Hormone
  n   Polyprotein → neuropeptides (peptide hormone ) conversion
2. Peptide Bond Isomerization (Intramolecular)
  n    Ser → esters
  n    Cys → thioesters
  n    Asp or Asn → isoaspartate
  n    Prolyl peptide cis-trans isomerization (prolyl isomerase
      catalyzed)
3. Peptide Bond Formation, Transpeptidation
  n   peptide bond splicing with peptide deletion and/or
      permutation Plant lectin – Concanvalin A
     B. Modifications Involving
        Amino and Carboxyl Termini
    • The N terminus:H3N+—
n    N-Formyl- (C1)          n   N-Aminoacyl-
n    N-Acetyl- (C2)          n   N-α-Ketoacyl-
n    N-Acyl- (C2, C4, C6,    n   N-Methyl-
     C8, C10)                n   N-Pyrrolidone
                                 carboxyl-
n    N-Lauroyl- (C12)
                             n   N-Glucuronyl-
n    N-Myristoyl- (C14)
                             n   N-Glycosyl-
n    N-Tetradeca (mono and
     di)enoyl- (C14:1;
     C14:2)
2. The C Terminus:
  n   Amide
  n   O-(ADP-ribosyl)-
  n   O-Methyl-
  n   -(N-Ethanolamine-glycan-phosphoinositides)
  n   -(Nα-TyT)
 C. Modifications Involving Individual
    Amino Acid (Side Chains)

• Arginine:
  n   Nω-(ADP-ribosyl)-   n   Ornithine
  n   Nω-Methyl-          n   Citrulline
  n   Nω-Dimethyl-        n   Nω-Phosphoryl-
  n   Nω-Nω’-Dimethyl-
2. Asparagine:
  n   N-Glycosyl-   n   Nε-(β-Aspartyl)lysine
  n   Aspartate     n   erythro-β-Hydroxy-
  n   N-Methyl-     n   N-(ADP-ribosyl)-
3. Aspartate:
  n   D-Asp (racemization)   n   β-Methylthio-
  n   β-Carboxy-             n   O-Phosphoryl-
  n   erythro-β-Hydroxy-     n   O-Methyl-
4. Cysteine:HS-CH2-
n   Cystine                                 n   S-Phycocyanobilin
n   S-γ-Glutamyl-                           n   S-p-Coumaroyl
n   S-(2-Histidyl)-                         n   S-(6-Flavin [FMN])
n   S-(3-Tyr)                               n   S-(8α-Flavin [FAD])
n   S-(sn-1-Glyceryl)-                      n   S-Coenzyme A
n   S-(sn-1-Diacylglyceryl)-                n   S-(ADP-ribosyl)-
n   S-(sn-1-{2,3,-Di-O-[3’ ,7’ ,11’ .15’-   n   S-Glycosyl-
    tetramethylhexadecyl]}glyceryl)-        n   Dehydroalanine
n   S-Palmitoyl-                            n   Lysinoalanine
n   S-Farnesyl-                             n   Lanthionine
n   S-Geranylgeranyl-                       n   Selenocysteine
n   S-Heme
5. Glutamate:
n   O-(ADP-ribosyl)
n   γ-Carboxy-
n   O-Methyl-
n   Nα-(γ-Glutamyl)-Glu1-5
n   Nα-(γ-Glutamyl)-Glu3-34
n   N- (γ-Glutamyl)ethanolaminephosphate)
n   S-γ-Glutamyl-Cys is listed under Cys
6. Glutamine:
  n   Glutamate
  n   Nε-(γ-Glutamyl)lysine
  n   N-(γ-Glutamyl)-L-ornithine
  n   N-(γ-Glutamyl)polyamine
  n   N,N-(Bis-γ-glutamyl)polyamine
  n   N5-Methyl-
7. Histidine:
  n   Diphthamide
  n   Nτ-(ADP-ribosyl)diphthamide
  n   N-Phosphoryl-
  n   Nπ-Methyl-
  n   4-Iodo-and diiodo-
  n   Nτ- and Nπ –(8α-flavin [FAD])
  n   Nπ-(8α-Flavin[FMN])
    8. Lysine:
n   Nε-Acetyl-                   n    Nε-Glycosyl-
n   Nε-( Nα-Monomethylalanyl)-   n    Nε-Mono-, di- , trimethyl-
n   Nε-Murein (peptidoglycan)    n    Hypusine:Nε-(4-amino-2-hydroxybutyl)-
n   Nε-Lipoyl-                   n    Allysine
n   Nε-Biotinyl-                 n   δ-Hydroxy-
n   Nε-Ubiquitinyl-              n   δ-Hydroxyallysine
n   Nε-Phosphoryl-               n   Cross-links
n   Nε-Phosphopyridoxyl-             (desmosines, syndesines, pyridinolines)
n   Nε-Retinyl-                  n   δ-Glyxosyloxy-
9. Methionine:
   n   Sulfoxide


10. Phenylalanine:
   n   β-Glycosyloxy-
11. Proline:
   n   3-Hydroxy-
   n   4-Hydroxy-
   n   3,4-Dihydroxy-
   n   O4-Arabinosylhydroxy-
   n   O4-Galactosylhydroxy-
   n   O4-Glucosylhydroxy-
12. Serine:HO-CH2
n   Selenocysteine               n   O-Glycosyl-
n   O-Phosphoryl-                n   Alanino(τ- or π-histidine)
n   O-Pantetheinephosphoryl-     n   Lanthionine
n   O-(GlcNAc-1-phosphoryl)-     n   O-Acetyl-
n   O-(Glycerol-1-phosphoryl)-   n   O-Fatty acyl-
n   O-Methyl-
How modifications are made ?
A. Nonenzymatic Reaction
n   deamidation:Asn, Gln
n   racemization:Asp, Ser
n   dehydroalanine:Cys, phosphor-Ser
n   slow oxidation:Cys, His, Met
n   slow cleavage and permutation of peptide bonds
n   reducing sugar reaction with NH2-group of aa’s or side
    chains (Lys):Maillard reaction (Browing reaction);
    Schiffs base reaction.
      B. Enzymatic Reaction

1. Irrversible, Unidirectional Reaction (permanently modified)
  n   N-linked glycosylation
  n   Carboxyl methylation
  n   S-isoprenylation-Cys
2. Irrversible, Bi-directional Reaction. (Signal Amplificaion)
  n   Phosphorylation (protein kinase) / Dephosphorylation
      (phosphatase):Ser, Tyr, Thr.

  n   Uridylyl and adenylyl transfer in bacterial glutamine
      synthetase
3. Reversible Reaction

   n    RS-SR + R’-SH ↹ R’-S-S-R + RSH(disulfide isomerase)
       Coupled with protein-folding process

				
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