1. Post-translational Modifications (PTM)
蛋白質體學
Proteomics 2016
Post-translational Modifications (PTM)
陳威戎 1

2. Classical Protein Biosynthesis
Proteins are synthesized in ribosomes and one trinucleotide specifies one amino acid.
Codons are universal and the starting codon (AUG) specifies Met or fMet.
Every protein should start with Met or fMet at the NH2-terminus.
Every protein should have no more than 20 amino acids.
However, many exceptional amino acids were found in many naturally occurring proteins, therefore, proteins must be modified before, during or after ribosomal protein synthesis.

3. Protein Biosynthesis at Three Levels of Modifications
20 Amino acids ＋ 20 tRNAs
Pre-translational
Modifications ↓
20 aa-tRNAs
Co-translational
Modifications ↓
Nascent polypeptide
Post-translational
Completed polypeptide

4. Examples of Three Levels of Protein Modifications
Levels Examples
1. Pre-translational a) Selenocysteine t-RNA
b) Nonnatural amino acid t-RNA
2. Co-translational a) Signal sequence cleavage
b) N-Glycosylation
3. Post-translational a) O-Glycosylation
b) Peptide bond cleavage
c) Protein splicing
d) Lipidation
e) Disulfide bond formation
f) Ubiquitination, Sumoylation

5. 以下內容感謝 “台灣大學 廖大修 名譽教授”、“長庚大學 游佳融 副教授”與
”台灣大學 張世宗 副教授” 提供

10. N-Acetylation Reactions

11. Acetylation Sites in Histones

12. Hyperacetylated Chromatin Domains
In eukaryotes, the genome is packaged into two general types of chromatin: heterochromatin, which appears compact or condensed throughout the cell cycle, and euchromatin, which appears condensed only prior to mitosis.
A small number of loci that exhibit covalent histone modifications by histone acetyltransferases (HAT), such as hyperacetylation.
The hyperacetylated domains occur exclusively at loci containing highly expressed, tissue-specific genes, and that they are involved in the activation of these genes.

13. Protein Acetylation in Prokaryotes
Protein acetylation plays a critical regulatory role in eukaryotes but prokaryotes also have the capacity to acetylate both the N-terminal residues and the side chain of Lys and is widespread for regulation of fundamental cellular processes.
Lys acetylation in particular can occur in proteins involved in transcription, translation, pathways associated with central metabolism and stress responses.
Like phosphorylation, acetylation appears to be an ancient reversible modification that can be present at multiple sites in proteins.
Acetylation is particularly important in regulating central metabolism in prokaryotes due to the requirement for acetyl-CoA and NAD+ for HAT and HDAC, respectively.

15. Methylase-Catalyzed Reactions

17. Protein Kinases and Their Preferred Substrate Specificities
Substrate recognition at the catalytic site involves specific residues in the region near the site of phosphorylation.

18. Protein Glycosylation Common in Eukaryotic Proteins

19. Sugar–Peptide Bonds

20. Sugar–Amino Acid Linkages of Glycoproteins
Type of bond Linkage Sugar Configuration Examples
N-glycosyl Asn GlcNAc β Ovalbumin, fetuin, insulin receptor
Asn Glc β Laminin, H. halobium S-layer
Asn GalNAc * H. halobium S-layer
Asn Rha * S. sanguis cell wall
Arg Glc β Sweet corn amylogenin
O-glycosyl Ser/Thr GalNAc α Mucins, fetuin, glycophorin
Ser/Thr GlcNAc β Nuclear and cytoplasmic proteins
Ser/Thr Gal α Earthworm collagen, B. cellulosoleum
Ser/Thr Man α Yeast mannoproteins
Ser/Thr Fuc α Coagulation and fibrinolytic factors
Ser/Thr Pse α C. jejuni flagellins
Ser Glc β Coagulation factors
Ser FucNAc β P. aeruginosa pili
Ser Xyl β Proteoglycans
Ser Gal α Cell walls of plants
Thr Man α M. tuberculosis secreted glycoproteins
Thr GlcNAc α Dictyosteliumh, T. cruzi
Thr Glc * Rho proteins (GTPases)
Thr Gal * H. halobium S-layer, vent worm collagen
Hyli Gal β Collagen, C1q complement
Hyp Ara β Potato lectin
Hyp Gal β Wheat endosperm
Hyp GlcNAc * Dictyostelium cytoplasmic proteins
Tyr Glc α Muscle and liver glycogenin
Tyr Glc β C. thermohydrosulfuricum S-layer
Tyr Gal β T. thermohydrosulfuricus S-layer
C-mannosylation Trp Man α RNase 2, interleukin 12, properdin
Phosphoglycosyl Ser GlcNAc α-1-P Dictyostelium proteinases
Ser Man α-1-P L. mexicana acid phosphatase
Ser Fuc β-1-P Dictyostelium proteins
Ser Xyl *-1-P T. cruzi cell surface
Glypiation Pr-C-(O)-EthN-6-P-Man T. brucei VSG, Thy-1, Sulfolobus proteins

21. Consensus Squences or Glycosylation Motifs for the Formation of Glycopeptide Bonds
Glycopeptide bond Consensus sequence or peptide domain
GlcNAc-β-Asn Asn-X-Ser/Thr (X = any amino acid except Pro)
Glc-β-Asn Asn-X-Ser/Thr
GalNAc-α-Ser/Thr Repeat domains rich in Ser, Thr, Pro, Gly, Ala in no special sequence
GlcNAc-α-Thr Thr rich domain near Pro residues
GlcNAc-β-Ser/Thr Ser/Thr rich domains near Pro, Val, Ala, Gly
Man-α-Ser/Thr Ser/Thr rich domains
Fuc-α-Ser/Thr EGF modules (Cys-X-X-Gly-Gly-Thr/Ser-Cys)
Glc-β-Ser EGF modules (Cys-X-Ser-X-Pro-Cys)
Xyl-β-Ser Ser-Gly (Ala) (in the vicinity of one or more acidic residues)
Glc/GlcNAc-Thr Rho: Thr-37d; Ras, Rac and Cdc42: Thr-35
Gal-Thr Gly-X-Thr (X = Ala, Arg, Pro, Hyp, Ser) (vent worm)
Gal-β-Hyl Collagen repeats (X-Hyl-Gly)
Ara-α-Hyp Repetitive Hyp rich domains (e.g., Lys-Pro-Hyp-Hyp-Val)
GlcNAc-Hyp Skp1: Hyp-143d
Glc-α-Tyr Glycogenin: Tyr-194d
GlcNAc-α-1-P-Ser Ser rich domains (e.g., Ala-Ser-Ser-Ala)
Man-α-1-P-Ser Ser rich repeat domains
Man-α-Trpf Trp-X-X-Trp
Man-6-P-EthN-C(O)-Pr GPI attached after cleavage of C-terminal peptide

26. Importance of Myristoylation
The myristate moiety participates in protein subcellular localization by facilitating protein-membrane interactions as well as protein-protein interactions.
Myristoylated proteins are crucial components of a wide variety of functions, including many signaling pathways, oncogenesis or viral replication.
Initially, myristoylation was described as a co-translational reaction that occurs after the removal of the initiator Met. It is now established that myristoylation can also occur post- translationally in apoptotic cells.
During apoptosis hundreds of proteins are cleaved by caspases and in many cases this cleavage exposes an N-terminal Gly within a cryptic myristoylation consensus sequence, which can be myristoylated.

27. Co- and Post-translational Attachment of Myristate to Proteins
Co-translational myristoylation: following removal of the initiator Met, the exposed N-terminal Gly is myristoylated.
Post-translational myristoylation: following cleavage of a cryptic myristoylation site by caspase cleavage, the exposed N- terminl Gly is myristoylated.

28. Biosynthesis of C-Terminal Isoprenyl Cysteine Methyl Ester
Proteins with a terminal Leu are modified by an isoprenyltransferase that transfers from geranylgeranyl pyrophosphate to Cys. Proteins with terminal residues, Ser, Ala, Met, or Gln are modified by another enzyme that adds farnesyl pyrophosphate to Cys.
Following the attachment of the isoprenyl moietis, the three terminal amino acids are cleaved by a protease.
Finally, an enzyme catalyzes the addition of a methyl group to the newly exposed carboxyl terminal Cys.

29. Isoprenyl Proteins and Their Functions
Isoprenyl proteins include many G-proteins, many isoprenyl proteins function in signal transduction processes across the plasma membrane or in the control of cell division.
The increased hydrophobicity of the C-terminus can lead to interactions with the membrane bilayer that result in membrane association of these proteins.
Alternatively, the isoprenyl and methyl groups may be specific targets for binding by other membrane "receptor" proteins, leading to a specific alignment of protein partners in signaling pathways.

30. S-Palmitoylation ︱
Structure: CH3(CH2)14CO-SCys
Protein S-palmitoylation is the thioester linkage of long-chain fatty acids to Cys in proteins.
Addition of palmitate to proteins facilitates their membrane interactions and trafficking, and it modulates protein-protein interactions and enzyme activity.
The reversibility of palmitoylation makes it a biological mechanism for regulating protein activity.
The regulation of palmitoylation occurs through the actions of acyltransferases and acylthioesterases. These molecules work in concert with thioesterases to regulate the palmitoylation status of numerous signaling molecules, ultimately influencing their function.

31. Functions of Palmitoylation
Similar to other lipid modifications, palmitoylation promotes membrane association of otherwise soluble proteins.
The function of palmitoylation, however, ranges beyond that of a simple membrane anchor.
Trafficking of lipidated proteins from the early secretory pathway to the plasma membrane is dependent upon palmitoylation in many cases.
Modification with fatty acids impacts the lateral distribution of proteins on the plasma membrane by targeting them to lipid rafts.
Palmitoylation also functions in the regulation of protein activity.

32. Glypiation
The process of adding glycosyl phosphatidyl inositol (GPI) to proteins, which has been termed glypiation, is carried out by a transamidase that cleaves the C-terminal peptide and concomitantly transfers the preassembled GPI anchor to the newly exposed carboxy- terminal amino acid residue to establish an amide bond between the latter and the ethanolamine moiety of the glycolipid.
GPI assembly takes place entirely on the cytoplasmic side of the ER and followed by its translocation to the lumenal side, where attachment to the protein takes place.
The transamidase reaction is carried out by a multiprotein complex that has as yet not been isolated in its intact form.
The carboxy-terminal signal peptide which is cleaved prior to binding of the GPI, consisting 15–30 amino acids, has structural similarities to the NH2-terminal peptide that functions in general to direct nascent chains into the ER lumen.

33. GPI Anchor
The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail.

35. Membrane-Associated Proteins in a Lipid Bilayer Containing Lipid Raft Domains
The GPI anchor anchors the modified protein in the outer leaflet of the cell membrane.

36. Functions of GPI-Anchored Proteins
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Biological role Protein Source
enzymes alkaline phosphatase mammalian tissues, Schistosoma
5′-nucleotidase mammalian tissues
dipeptidase pig and human kidney, sheep lung
cell-cell interaction LFA human blood cells
PH guinea pig sperm
complement regulation CD55 (DAF) human blood cells
CD human blood cells
mammalian antigens Thy mammalian brain and lymphocytes
Qa mouse lymphocytes
CD human monocytes
CD human lymphocytes
protozoan antigens VSG T. brucei
1G T. cruzi
procyclin T. brucei
miscellaneous scrapie prion protein hamster brain
CD16b human neutrophils
folate-binding protein human epithelial cells

39. ■ Steps involved in protein degradation
20S Core
6 ATPases
Peptidases
19S
Poly-ubiquitin chain
Antigenic
peptides
Amino acids
2-25 residues
Cytosolic
peptidases

40. ■ The ubiquitination cascade
Degradation

42. ■ Comparison of ubiquitin and SUMO
Small Ubiquitin-like Modifier (SUMO)
C-terminal GG motif

43. Sumoylation Processing Desumoylation
■ The mechanism of reversible sumoylation
SENP: sentrin- specific protease
Sumoylation E1
Processing E2
Desumoylation E3

44. More active or inactive
■ Molecular consequences of sumoylation
Conformational
change
Interfere interaction
Provide a binding site
More active or inactive

46. Cell division Oncogenesis Signal transduction Stress response
■ SUMO participates in diverse cellular processes
DNA damage repair
Chromosome segregation
Nuclear transport
Cell division
Signal transduction
Hypoxia
Stress response
Inflammatory response
Oncogenesis
Flowering time in plants

48. Activation of Proteases
After trypsinogen enters the small intestine, it is converted into its active form, trypsin by enteropeptidase.
Now trypsin hydrolyzes more trypsinogen and starts to hydrolyze chymotrpsinogen to active their forms.

64. Proteomic analysis of PTMs
Mann and Jensen, Nature Biotech. 21, 255 (2003)