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蛋白質體學
Proteomics 2014
Post-translational
Modifications (PTM)
陳威戎
2014.12.15
Classical Protein Biosynthesis
1. Proteins are synthesized in ribosomes and one
trinucleotide specifies one amino acid.
2. Codons are universal and the starting codon (AUG)
specifies Met or fMet.
3. Every protein should start with Met or fMet at the
NH2-terminus.
4. 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.
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
Modifications
↓
Completed polypeptide
Examples of Three Levels of Protein
Modifications
Levels
1. Pre-translational
Examples
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
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
N-Acetylation Reactions
Acetylation Sites in Histones
Hyperacetylated Chromatin Domains
1.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.
2.A small number of loci that exhibit covalent histone
modifications by histone acetyltransferases (HAT), such
as hyperacetylation.
3.The hyperacetylated domains occur exclusively at loci
containing highly expressed, tissue-specific genes, and that
they are involved in the activation of these genes.
Protein Acetylation in Prokaryotes
1. 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.
2. Lys acetylation in particular can occur in proteins involved
in transcription, translation, pathways associated with
central metabolism and stress responses.
3. Like phosphorylation, acetylation appears to be an ancient
reversible modification that can be present at multiple
sites in proteins.
4. Acetylation is particularly important in regulating central
metabolism in prokaryotes due to the requirement for
acetyl-CoA and NAD+ for HAT and HDAC, respectively.
Methylase-Catalyzed Reactions
Protein Kinases and Their Preferred Substrate
Specificities
Substrate recognition at the catalytic site involves specific residues
in the region near the site of phosphorylation.
Protein Glycosylation
Common in Eukaryotic Proteins
Sugar–Peptide Bonds
Sugar–Amino Acid Linkages of Glycoproteins
Type of bond
N-glycosyl
O-glycosyl
C-mannosylation
Phosphoglycosyl
Glypiation
Linkage
Sugar
Configuration
Asn
GlcNAc
Asn
Glc
Asn
GalNAc
Asn
Rha
Arg
Glc
Ser/Thr
GalNAc
Ser/Thr
GlcNAc
Ser/Thr
Gal
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
Pse
Ser
Glc
Ser
FucNAc
Ser
Xyl
Ser
Gal
Thr
Man
Thr
GlcNAc
Thr
Glc
Thr
Gal
Hyli
Gal
Hyp
Ara
Hyp
Gal
Hyp
GlcNAc
Tyr
Glc
Tyr
Glc
Tyr
Gal
Trp
Man
Ser
GlcNAc
Ser
Man
Ser
Fuc
Ser
Xyl
Pr-C-(O)-EthN-6-P-Man
β
β
*
*
β
α
β
α
α
α
α
β
β
β
α
α
α
*
*
β
β
β
*
α
β
β
α
α-1-P
α-1-P
β-1-P
*-1-P
Examples
Ovalbumin, fetuin, insulin receptor
Laminin, H. halobium S-layer
H. halobium S-layer
S. sanguis cell wall
Sweet corn amylogenin
Mucins, fetuin, glycophorin
Nuclear and cytoplasmic proteins
Earthworm collagen, B. cellulosoleum
Yeast mannoproteins
Coagulation and fibrinolytic factors
C. jejuni flagellins
Coagulation factors
P. aeruginosa pili
Proteoglycans
Cell walls of plants
M. tuberculosis secreted glycoproteins
Dictyosteliumh, T. cruzi
Rho proteins (GTPases)
H. halobium S-layer, vent worm collagen
Collagen, C1q complement
Potato lectin
Wheat endosperm
Dictyostelium cytoplasmic proteins
Muscle and liver glycogenin
C. thermohydrosulfuricum S-layer
T. thermohydrosulfuricus S-layer
RNase 2, interleukin 12, properdin
Dictyostelium proteinases
L. mexicana acid phosphatase
Dictyostelium proteins
T. cruzi cell surface
T. brucei VSG, Thy-1, Sulfolobus proteins
Consensus Squences or Glycosylation Motifs for the
Formation of Glycopeptide Bonds
Glycopeptide bond
GlcNAc-β-Asn
Glc-β-Asn
GalNAc-α-Ser/Thr
GlcNAc-α-Thr
GlcNAc-β-Ser/Thr
Man-α-Ser/Thr
Fuc-α-Ser/Thr
Glc-β-Ser
Xyl-β-Ser
Glc/GlcNAc-Thr
Gal-Thr
Gal-β-Hyl
Ara-α-Hyp
GlcNAc-Hyp
Glc-α-Tyr
GlcNAc-α-1-P-Ser
Man-α-1-P-Ser
Man-α-Trpf
Man-6-P-EthN-C(O)-Pr
Consensus sequence or peptide domain
Asn-X-Ser/Thr (X = any amino acid except Pro)
Asn-X-Ser/Thr
Repeat domains rich in Ser, Thr, Pro, Gly, Ala in no special sequence
Thr rich domain near Pro residues
Ser/Thr rich domains near Pro, Val, Ala, Gly
Ser/Thr rich domains
EGF modules (Cys-X-X-Gly-Gly-Thr/Ser-Cys)
EGF modules (Cys-X-Ser-X-Pro-Cys)
Ser-Gly (Ala) (in the vicinity of one or more acidic residues)
Rho: Thr-37d; Ras, Rac and Cdc42: Thr-35
Gly-X-Thr (X = Ala, Arg, Pro, Hyp, Ser) (vent worm)
Collagen repeats (X-Hyl-Gly)
Repetitive Hyp rich domains (e.g., Lys-Pro-Hyp-Hyp-Val)
Skp1: Hyp-143d
Glycogenin: Tyr-194d
Ser rich domains (e.g., Ala-Ser-Ser-Ala)
Ser rich repeat domains
Trp-X-X-Trp
GPI attached after cleavage of C-terminal peptide
Importance of Myristoylation
1.The myristate moiety participates in protein subcellular
localization by facilitating protein-membrane interactions
as well as protein-protein interactions.
2.Myristoylated proteins are crucial components of a wide
variety of functions, including many signaling pathways,
oncogenesis or viral replication.
3.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.
4.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.
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 Nterminl Gly is myristoylated.
Biosynthesis of C-Terminal Isoprenyl Cysteine Methyl Ester
1. 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.
2. Following the attachment of the isoprenyl moietis, the three terminal
amino acids are cleaved by a protease.
3. Finally, an enzyme catalyzes the addition of a methyl group to the
newly exposed carboxyl terminal Cys.
Isoprenyl Proteins and Their Functions
1.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.
2.The increased hydrophobicity of the C-terminus can lead
to interactions with the membrane bilayer that result in
membrane association of these proteins.
3.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.
S-Palmitoylation
︱
Structure: CH3(CH2)14CO-SCys
︱
1. Protein S-palmitoylation is the thioester linkage of long-chain fatty
acids to Cys in proteins.
2. Addition of palmitate to proteins facilitates their membrane
interactions and trafficking, and it modulates protein-protein
interactions and enzyme activity.
3. The reversibility of palmitoylation makes it a biological mechanism
for regulating protein activity.
4. 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.
Functions of Palmitoylation
1.Similar to other lipid modifications, palmitoylation
promotes membrane association of otherwise soluble
proteins.
2.The function of palmitoylation, however, ranges beyond
that of a simple membrane anchor.
3.Trafficking of lipidated proteins from the early secretory
pathway to the plasma membrane is dependent upon
palmitoylation in many cases.
4.Modification with fatty acids impacts the lateral
distribution of proteins on the plasma membrane by
targeting them to lipid rafts.
5.Palmitoylation also functions in the regulation of protein
activity.
Glypiation
1. 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 carboxyterminal amino acid residue to establish an amide bond between the
latter and the ethanolamine moiety of the glycolipid.
2. 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.
3. The transamidase reaction is carried out by a multiprotein complex that
has as yet not been isolated in its intact form.
4. 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.
GPI Anchor
The GPI anchor is a complex
structure comprising a
phosphoethanolamine linker,
glycan core, and phospholipid tail.
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.
Biosynthesis of GPI Anchor
In trypanosomes
In mammalian cells
PI, phophatidylinositol; G, glucosamine;
Ac, acetyl; M, mannose; pE,
Phosphoehanolamine; (acyl), fatty acid
linked to inositol.
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-3
human blood cells
PH-20
guinea pig sperm
complement regulation
CD55 (DAF)
human blood cells
CD59
human blood cells
mammalian antigens
Thy-1
mammalian brain and lymphocytes
Qa-2
mouse lymphocytes
CD14
human monocytes
CD52
human lymphocytes
protozoan antigens
VSG
T. brucei
1G7
T. cruzi
procyclin
T. brucei
miscellaneous
scrapie prion protein
hamster brain
CD16b
human neutrophils
folate-binding protein
human epithelial cells
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■ Steps involved in protein degradation
19S
20S Core
Peptidases
6 ATPases
Poly-ubiquitin chain
2-25 residues
Antigenic
peptides
Cytosolic
peptidases
Amino acids
■ The ubiquitination cascade
E2
E1
E3
Degradation
■ Comparison of ubiquitin and SUMO
Small Ubiquitin-like Modifier (SUMO)
C-terminal GG motif
■ The mechanism of reversible sumoylation
SENP: sentrin- specific protease
Sumoylation
Processing
E1
E2
Desumoylation
E3
■ Molecular consequences of sumoylation
Interfere interaction
Provide a binding site
Conformational
change
More active or inactive
■ SUMO participates in diverse cellular processes
DNA damage repair Chromosome segregation
Nuclear transport
Cell division
Hypoxia
Stress response
Inflammatory response
Oncogenesis
Flowering time in plants
Activation of Proteases
1. After trypsinogen enters the small
intestine, it is converted into its
active form, trypsin by
enteropeptidase.
2. Now trypsin hydrolyzes more
trypsinogen and starts to
hydrolyze chymotrpsinogen to
active their forms.
Proteomic analysis of PTMs
Mann and
Jensen, Nature
Biotech. 21,
255 (2003)