Translation

and
Post Translation
Modifications

Dr. Bonuke Anyona

Translation
Amino
Polypeptide acids

tRNA with
amino acid
attached
Ribosome

tRNA
C
G
Anticodon
U G G U U U G G C

5 Codons 3
mRNA
 Learning Objectives
 By the end of this Topic, you should be able
to:
i. Describe the process of building a polypeptide
(Translation)
ii. Explain the structure and role of ribosomes in
the translation process
iii. Describe the targeting of resulting polypeptide
 Translation

 Is the RNA-directed synthesis of a

polypeptide
 Genetic information flows from mRNA to

protein through the process of translation.

 Molecular Components of
Translation
 A cell translates a mRNA message into
protein with the help of transfer RNA
(tRNA)
 tRNA transfer amino acids to the growing

polypeptide in a ribosome
 Translation is a complex process in terms of

its biochemistry and mechanics

Translation:
The basic
concept
Amino
Polypeptide acids

tRNA with
amino acid
attached
Ribosome
Trp

Phe Gly

tRNA
C
C
C G
A
C C
G
Anticodon
A A A

U G G U U U G G C

5 Codons 3
mRNA
 The Structure and Function of
Transfer RNA (tRNA)

 Molecules of tRNA are not identical

 Each carries a specific amino acid on one end
 Each has an anticodon on the other end; the

anticodon base-pairs with a complementary codon

on mRNA.
 A tRNA molecule consists of a single RNA strand
that is about 80 nucleotides long.
 Flattened into one plane to reveal its base

pairing, a tRNA molecule looks like a cloverleaf.

 The Structure of tRNA
3
Amino acid
attachment
site
5
Amino acid
attachment
5 site
3

Hydrogen
bonds
Hydrogen
bonds

A A G
3 5
Anticodon Anticodon
Anticodon
(c) Symbol used
(a) Two-dimensional structure (b) Three-dimensional structure
 The Structure of tRNA
 Because of hydrogen bonds, tRNA twists and
folds into a three-dimensional molecule.
 tRNA is roughly L-shaped.

 Accurate translation requires two steps:

 First: a correct match between a tRNA and an
amino acid, done by the enzyme aminoacyl-tRNA
synthetase.
 Second: a correct match between the tRNA
anticodon and a mRNA codon.
 Flexible pairing at the third base of a codon is
called wobble and allows some tRNAs to bind
to more than one codon.
 Aminoacyl-tRNA
synthetase (enzyme)

Amino acid

P P P Adenosine

ATP

An aminoacyl-tRNA synthetase joining a

specific amino acid to a tRNA
 Aminoacyl-tRNA
synthetase (enzyme)

Amino acid

P Adenosine

P P P Adenosine P Pi

ATP
Pi
P
i

An aminoacyl-tRNA synthetase joining a

specific amino acid to a tRNA
 Aminoacyl-tRNA
synthetase (enzyme)

Amino acid

P Adenosine

P P P Adenosine P Pi

ATP Aminoacyl-tRNA
Pi
P
i tRNA synthetase

tRNA

Amino
acid

P Adenosine

AMP
Computer model

An aminoacyl-tRNA synthetase joining a

specific amino acid to a tRNA
 Aminoacyl-tRNA
synthetase (enzyme)

Amino acid

P Adenosine

P P P Adenosine P Pi

ATP Aminoacyl-tRNA
Pi
P
i tRNA synthetase

tRNA

Amino
acid

P Adenosine

AMP
Computer model

Aminoacyl tRNA
(“charged tRNA”)

An aminoacyl-tRNA synthetase joining a

specific amino acid to a tRNA
 Ribosomes
 Ribosomes facilitate specific coupling of tRNA
anticodons with mRNA codons in protein
synthesis.
 The two ribosomal subunits (large and small) are

made of proteins and ribosomal RNA (rRNA).

 Bacterial and eukaryotic ribosomes are
somewhat similar but have significant
differences: some antibiotic drugs specifically
target bacterial ribosomes without harming
eukaryotic ribosomes.
The anatomy of a functioning
ribosome Growing
polypeptide Exit tunnel
tRNA
molecules

Large
subunit
E P
A

Small
subunit

5
mRNA 3

(a) Computer model of functioning ribosome

Growing polypeptide
P site (Peptidyl-tRNA Amino end
Exit tunnel Next amino
binding site)
acid to be
added to
A site (Aminoacyl- polypeptide
tRNA binding site) chain
E site
(Exit site)
E tRNA
E P A Large
mRNA 3
subunit

mRNA
binding site Small 5 Codons
subunit
(b) Schematic model showing binding sites (c) Schematic model with mRNA and tRNA
 Large
subunit
E P

Ribosomes A

Small
subunit

5
mRNA
A 3
ribosome has three
binding
(a) Computer model of functioning ribosome sites for tRNA
Growing polypeptide
P site (Peptidyl-tRNA
Exit tunnel
 The P site holds tRNA that
Amino end
binding site) Next amino
carries the acid to be growing
added to
A site (Aminoacyl-
tRNA binding site)
polypeptide chain.polypeptide
E site chain
(Exit site)  The A site holds the tRNA
E tRNA
E P A Large
subunit
that carries the next
mRNA 3 amino
mRNA acid to be added to the chain.
binding site Small 5 Codons
subunit  The E site is the exit site,
(b) Schematic model showing binding sites (c) Schematic model with mRNA and tRNA
where discharged tRNAs
leave the ribosome.
 Building a Polypeptide

 The three stages of translation

 Initiation
 Elongation

 Termination

 All three stages require protein “factors”

that aid in the translation process.
Ribosome Association and Initiation
of Translation
 The initiation stage of translation brings
together mRNA, a tRNA with the 1st amino
acid, and two ribosomal subunits.
 First, a small ribosomal subunit binds with

mRNA and a special initiator tRNA.

 Then the small subunit moves along the mRNA

until it reaches start codon (AUG).

 Proteins called initiation factors bring in large

subunit that completes the translation

initiation complex.
 The Initiation of
Translation
Large
ribosomal
subunit

3 U A C 5 P site
Met 5 A U G 3 Met

Pi
Initiator 
tRNA GTP GDP
E A
mRNA
5 5
3 3
Start codon
Small
mRNA binding site ribosomal Translation initiation complex
subunit
 Elongation of Polypeptide
Chain
 During the elongation stage, amino acids are
added one by one to the preceding amino acid
at the C-terminus of the growing chain.
 Each addition involves proteins called
elongation factors and occurs in three steps:
codon recognition, peptide bond formation, and
translocation.
 Translation proceeds along the mRNA in a 5′ to

3′ direction.
 Amino end of
polypeptide

E
mRNA 3
P A
site site
5

The Elongation Cycle of

Translation
 Amino end of
polypeptide

E
mRNA 3
P A
site site GTP
5
GDP  P i

P A

The Elongation Cycle of

Translation
 Amino end of
polypeptide

E
mRNA 3
P A
site site GTP
5
GDP  P i

P A

P A

The Elongation Cycle of

Translation
 Amino end of
polypeptide

E
mRNA 3
Ribosome ready for P A
next aminoacyl tRNA site site GTP
5
GDP  P i

E E

P A P A

GDP  P i

GTP

P A

The Elongation Cycle of

Translation
 Termination of Translation
 Termination occurs when a stop codon in the
mRNA reaches the A site of the ribosome.
 The A site accepts a protein called a release

factor.
 The release factor causes the addition of a

water molecule instead of an amino acid

 This reaction releases the polypeptide, and the

translation assembly then comes apart.

 The termination of
translation
Release
factor

3

5

Stop codon
(UAG, UAA, or UGA)
 Release
factor Free
polypeptide

3 3
2 GTP
5 5
2 GDP  2 P
Stop codon i

(UAG, UAA, or UGA)

 Release
factor Free
polypeptide

5
3 3
2 GTP 3
5 5
2 GDP  2 P
Stop codon i

(UAG, UAA, or UGA)

 Polyribosomes
 A number of ribosomes can Growing
Completed
polypeptide

translate a single mRNA Incoming

ribosomal
polypeptides

simultaneously, forming a subunits

Start of
End of
mRNA

polyribosome (or polysome).

mRNA
(5 end)
(a) (3 end)

 Polyribosomes enable a cell to Ribosomes

mRNA

make many copies of a

polypeptide very quickly (b)
0.1 m
 Completing and Targeting the
Functional Protein
 Often translation is not sufficient to make a
functional protein.

 Polypeptide chains are modified after

translation or targeted to specific sites in the
cell.
 Protein Folding and Post-
Translational Modifications
 During and after synthesis, a polypeptide
chain spontaneously coils and folds into its
three-dimensional shape.
 Proteins may also require post-translational

modifications before full functionality.

 Some polypeptides are activated by enzymes

that cleave them.

 Other polypeptides come together to form the

subunits of a protein.
 Targeting Polypeptides to
Specific Locations
 Two populations of ribosomes are evident in
cells: free ribsomes (in the cytosol) and bound
ribosomes (attached to the ER).
 Free ribosomes mostly synthesize proteins that

function in the cytosol.

 Bound ribosomes make proteins of the
endomembrane system and proteins that are
secreted from the cell.
 Ribosomes are identical and can switch from

free to bound.
 Targeting Polypeptides to Specific
Locations
 Polypeptide synthesis always begins in the cytosol
 Synthesis finishes in the cytosol unless the
polypeptide signals the ribosome to attach to the
ER.
 Polypeptides destined for the ER or for secretion

are marked by a signal peptide.

 A signal-recognition particle (SRP) binds to the

signal peptide.
 The SRP brings the signal peptide and its

ribosome to the ER.

 Protein
Post-translational
Modification
 Learning Objectives
 By the end of this Topic, you should be able
to:
i. Describe the post translation modifications
ii. Explain types of post translation modifications
iii. Describe diseases/syndromes associated with
errors in post translation modifications
 Post-translational modification
(PTM)

 What is it ?
Addition of groups or deletion of parts to
make a finished functional protein

 What groups ? How much ? Where ?

- methyl
- acetyl
- glyco
- phospho
 Post-translational modification
(PTM)

 What purpose ?
- targeting (eg. some lipoproteins)
- stability (eg. secreted glycoproteins )
- function (eg. surface glycoproteins)
- control of activity (eg. clotting factors,
caspases)
 Post-translational modification
(PTM)

Chemical modifications that take place at
certain amino acid residues after the protein is
synthesized by translation are known as post-
translational modifications.

These are essential for normal functioning of
the protein.

Some of the most commonly observed PTMs
include: Phosphorylation, Glycosylation,
Acylation, Alkylation and Hydroxylation.
a) Phosphorylation: The process by which a
phosphate group is attached to certain amino
acid side chains in the protein, most
commonly serine, threonine and tyrosine.

b) Glycosylation: The attachment of sugar

moieties to nitrogen or oxygen atoms present
in the side chains of amino acids like
aspargine, serine or threonine.
c) Acylation: The process by which an acyl
group is linked to the side chain of amino
acids like aspargine, glutamine or lysine.

d) Alkylation: Addition of alkyl groups, most

commonly a methyl group to amino acids such
as lysine or arginine. Other longer chain alkyl
groups may also be attached in some cases.
e) Hydroxylation: This PTM is most often
found on proline and lysine residues which
make up the collagen tissue.
It enables crosslinking and therefore
strengthening of the muscle fibres.
 Definition of Terms
Protein translation: The process by which the
mRNA template is read by ribosomes to
synthesize the corresponding protein molecule
on the basis of the three letter codons, which
code for specific amino acids.

Cytosol: A cellular compartment that serves as

the site for protein synthesis
 Definition of Terms
Signal sequence: A sequence that helps in
directing the newly synthesized polypeptide
chain to its appropriate intracellular organelle.
This sequence is most often cleaved following
protein folding and PTM.

Endoplasmic reticulum: A membrane-bound

cellular organelle that acts as a site for post-
translational modification of the newly
synthesized polypeptide chains.
Definition of Terms
Cleaved protein: The protein product obtained
after removal of certain amino acid sequences
such as N- or C-terminal sequences, signal
sequence etc.
 CENTRAL DOGMA
Gene encoding region (ORF)
↓transcription
mRNA
↓translation
Protein (nascent protein, precursor protein)
↓protein processing, post-translational
modification
Mature protein
↓folding
Biological active protein
Process of post-translational modification

Translated
mRNA Protein
Cytosol Ribosome

P
Endoplasmic P
reticulum (ER)
Protein folding &
PTMs
Removal of CH3 CH3
certain N- and C- Cleaved protein
terminal residuesProtease
Glc
Glc

Description of the action

Once the protein has been synthesized by the ribosome from its corresponding mRNA in the cytosol,
many proteins get directed towards the endoplasmic reticulum for further modification. Certain N
and C terminal sequences are often cleaved in the ER after which they are modified by various
enzymes at specific amino acid residues. These modified proteins then undergo proper folding to give
the functional protein.
Increased complexity of proteome due to PTMs
A A C G G U G C C G U G C A C G C A C U A C G C A C U

Gene sequence
Expected protein Actual protein
structure structure

Glc
P
Ala CH3
Asn His Ala H is
Gly Ar Asn
Val Ala Thr G ly Ar
Val Ala Thr
Leu g
Leu g

Description of the action

The final structure of functional proteins most often does not correlate directly with
the corresponding gene sequence. This is due to PTMs that occur at various amino
acid residues in the protein, which cause changes in interactions between amino acid
side chains thereby modifying protein structure. This further increases complexity of
the proteome as compared to the genome.
Phosphorylation reactions
COO- COO-
Kinase
H C R OH H C R O PO43-

NH3+ ATP ADP NH3+

Amino acid Phosphorylated
residue residue

CH2 Ser
CH
Thr
R
CH3

CH2 Tyr

Description of the action

Phosphorylation of amino acid residues is carried out by kinases that most commonly modify side chains
of amino acids containing a hydroxyl group. Phosphorylation requires the presence of a phosphate donor
molecule such as ATP, GTP or other phoshorylated substrates. Serine is the most commonly
phosphorylated residue followed by threonine and tyrosine. Removal of phosphate groups is carried out
by the phosphatase enzyme and thus this forms one of the most important mechanisms for regulation of
proteins.
Glycosylation reactions
N-linked Glycosylation
COO- Sugar residues
COO -
Glycosyl
H C CH2 CO
H C CH2 CONH2 transferase
N
NH3+
NH3 +

Asn N-linked amino acid

O-linked Glycosylation
COO-
COO-
Glycosyl H C R O
H C R OH
transferase
NH3+
NH3+
Ser/Thr
O-linked amino acid

Description of the action

Glycosylation involves enzymatic addition of saccharide molecules to amino acid side chains. This can be
of two types: (a) N-linked glycosylation, which links sugar residues to amide group of aspargine and (b)
O-linked glycosylation, which links sugar moieties to hydroxyl groups of serine or threonine. Suitable
glycosyl transferase enzymes catalyze these reactions. Sugar residues that are attached most commonly
include galactose, mannose, glucose, N-acetylglucosamine, N-acetylgalactosamie as well as fucose.
Protein Glycosylation

 The most important and complex form of PTM

 Approx. 1% mammalian genes

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
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 glycosyltransferases to 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
The ABO(H) blood group determinant is an
O-linked polysaccharide
•In 1901, Karl
Landsteiner discovered
blood group antigens.
•Since that time nearly
200 antigens have been
identified.
•Carbohydrate-
dependant blood group
antigens are carried by
both glycoproteins and
glycolipids. Gal 1-3 capping, Fuc 1-3 on
inner GlcNAc; blood group
determinants
 Protein Glycosylation - Biological
Significance
 Oligosaccharides may be a tissue-specific marker

 Carbohydrates may alter the polarity and solubility

 Steric interaction between protein and oligosaccharides

dictates certain protein 3D structure

 The bulkiness and negative charge of oligosaccharide

chain may protect protein from the attack by proteolytic
enzymes
 The Sugar Code
Carbohydrates as Informational Molecule

 Information: intracellular targeting of proteins,

cell-cell interactions, tissue development,
extracellular signals
 Improved methods for structural analysis
 Sugar code - The unique complex structure
of oligosaccharide on glycoprotein read by protein
 Lectins
carbohydrate-binding proteins

 Lectins read sugar code and mediate many biological

processes :

[1] Cell-cell recognition

[2] Signaling
[3] Adhesion
[4] Intracellular targeting of newly synthesized
proteins
Role of oligosaccharides in recognition and
adhesion
 Post-translational Modification
Numerous and diverse
 Adenylation
 Cleavage of signal peptides
 Sulfonation
 Phosphorylation
 Prenylation
 Amidation
 Myristoylation
 Glycosylation
 Acylation
 Hydroxylation
 Acetylation
 Ubiquitination
 Methylation
 Addition of prosthetic groups
 Oxidative crosslinking
 Iodination
 N-Glutamyl cyclization
 Carboxylation

 Change the charge, conformation or size of

protein molecule
Effects of Post-translational Modification
 stability of protein

 biochemical activity (activity regulation)

 protein targeting (protein localization)

 protein signaling (protein-protein

interaction,cascade amplification)
 Posttranslational Modification
Modification Charge-dependent change
Acylation loss of a-amino positive charge
Alkylation alteration of a- or e-amino positive
group
Carboxylmethylation esterification of specific carboxyl group
Phoshorylation mainly modify Ser, Thr and Tyr
Sulfation mainly modify Tyr
Carboxylation introduce negative charge
Sialyation mainly on Asn, Thr and Ser
Proteolytic processing truncation leads to change of pI
 Posttranslational Modification
Location Modification

Nucleus acetylation, phosphorylation

Lysosome mannose-6-phosphate labelled N-linked sugar
Mitochondria N-formyl acylation
Golgi N- and O-linked ologosaccharide, sulfation,
palimitoylation
ER N-linked oligosaccharide, GPI-anchor
Cytosol acetylation, methylation, phosphorylation,
Ribosome myristoylation
Plasma membrane N- and O-glycosylation, GPI-anchor
Extraceullar fluid N- and O-glycosylation, acetylation,
phosphorylation
Extrallular matrix N- and O-glycosylation, phosphorylation,
hydroxylation
Posttranslational Modification
Examples:

 Chromatin Structure/function - acetylation

 Regulation of mitochondrial processes –
phosphorylation
 Evade immune system – glycosylation
 Gene regulation – glycosylation
 Recognition - glycosylation
Histone and Nucleosome Function

 The nucleosome not only serves to compact the

genetic material but also provides information that
affects nuclear functions including DNA replication,
repair and transcription.

 This information is conveyed through numerous

combinations of histone post-translational
modifications (PTMs) and histone variants.

 How and when these combinations of PTMs are

imposed and to what extent they are determined by
the choice of a specific histone variant.
 Dynamic Change of Chromatin Structure
 Structural changes in chromatin are facilitated by a
variety of nuclear activities that reversibly modify
nucleosomes and nucleosome-remodeling complexes
- Such as histone kinases, methylases, acetylases,
histone deacetylases, DNA methylases

 The nucleus also contains numerous proteins, such as

the high mobility group N (HMGN) proteins, which
bind to DNA and to nucleosomes and induce structural
changes that affect transcription, replication and other
DNA-dependent activities
Chromatin Remodeling
 The regulated alteration of chromatin
structure, can be accomplished by :
(1) covalent modification of histones
(2) action of ATP-dependent remodeling
complexes.

 A variety of mechanisms can be used to

remodel chromatin; some act locally on a
single nucleosome and others act more
broadly.
 H3 Barcode Hypotheses
 Histones 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 structure into
activated or repressed transcriptional state
(2) acting as a docking site for transcriptional
regulators
Types of Post-translational
Modification
A. Modification Involving Peptide
Bonds Cleavage
(limited proteolysis)
1. 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
2. Peptide Bond Isomerization (Intramolecular)

 Ser → esters
 Cys → thioesters
 Asp or Asn → isoaspartate
 Prolyl peptide cis-trans isomerization (prolyl
isomerase catalyzed)

3. Peptide Bond Formation, Transpeptidation

 peptide bond splicing with peptide deletion and/or
permutation Plant lectin – Concanvalin A
B. Modifications Involving
Amino and Carboxyl Termini
1. The N terminus ： H3N+—
 N-Formyl- (C1)  N-Aminoacyl-
 N-Acetyl- (C2)  N-α-Ketoacyl-
 N-Acyl- (C2, C4, C6,  N-Methyl-
C8, C10)  N-Pyrrolidone
 N-Lauroyl- (C12) carboxyl-
 N-Glucuronyl-
 N-Myristoyl- (C14)
 N-Glycosyl-
 N-Tetradeca (mono and
di)enoyl- (C14 ： 1 ；
C14 ： 2)
2.The C Terminus ：

 Amide
 O-(ADP-ribosyl)-
 O-Methyl-
 -(N-Ethanolamine-glycan-phosphoinositides)
 -(Nα-TyT)
C. Modifications Involving Individual
Amino Acid (Side Chains)
1. Arginine ：
 Nω-(ADP-ribosyl)-  Ornithine
 Nω-Methyl-  Citrulline
 Nω-Dimethyl-  Nω-Phosphoryl-
 Nω-Nω’-Dimethyl-

2. Asparagine ：
 N-Glycosyl-  Nε-(β-Aspartyl)lysine
 Aspartate  erythro-β-Hydroxy-
 N-Methyl-  N-(ADP-ribosyl)-
3. Aspartate ：
 D-Asp (racemization)  β-Methylthio-
 β-Carboxy-  O-Phosphoryl-
 erythro-β-Hydroxy-  O-Methyl-

4. Cysteine ： HS-CH2-
 Cystine
 S-γ-Glutamyl-
 S-Phycocyanobilin
 S-(2-Histidyl)-
 S-p-Coumaroyl
 S-(3-Tyr)
 S-(6-Flavin [FMN])
 S-(sn-1-Glyceryl)-
 S-(8α-Flavin [FAD])
 S-(sn-1-Diacylglyceryl)-
 S-Coenzyme A
 S-(sn-1-{2,3,-Di-O-[3’ ,7’ ,11’ .15’-
 S-(ADP-ribosyl)-
tetramethylhexadecyl]}glyceryl)-
 S-Glycosyl-
 S-Palmitoyl-
 Dehydroalanine
 S-Farnesyl-
 Lysinoalanine
 S-Geranylgeranyl-
 Lanthionine
 S-Heme
 Selenocysteine
5. 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

6. Glutamine ：
 Glutamate
 Nε-(γ-Glutamyl)lysine
 N-(γ-Glutamyl)-L-ornithine
 N-(γ-Glutamyl)polyamine
 N,N-(Bis-γ-glutamyl)polyamine
 N5-Methyl-
7. Histidine ：
 Diphthamide
 Nτ-(ADP-ribosyl)diphthamide
 N-Phosphoryl-
 Nπ-Methyl-
 4-Iodo-and diiodo-
 Nτ- and Nπ –(8α-flavin [FAD])
 Nπ-(8α-Flavin[FMN])

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

10. Phenylalanine ：
 β-Glycosyloxy-

11. Proline ：
 3-Hydroxy-
 4-Hydroxy-
 3,4-Dihydroxy-
 O4-Arabinosylhydroxy-
 O4-Galactosylhydroxy-
 O4-Glucosylhydroxy-
12. Serine ： HO-CH2

 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-Fatty acyl-
How modifications are
made ?
A. Nonenzymatic Reaction

 deamidation ： Asn, Gln

 racemization ： Asp, Ser
 dehydroalanination ： 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.
B. Enzymatic Reaction
1. Irreversible, Unidirectional Reaction
(permanently modified)
 N-linked glycosylation
 Carboxyl methylation
 S-isoprenylation-Cys

2. Irréversible, Bi-directional Reaction

(Signal Amplificaion)
 Phosphorylation (protein kinase) / Dephosphorylation
(phosphatase) ： Ser, Tyr, Thr.
 Uridylyl and adenylyl transfer in bacterial glutamine
synthetase
3. Reversible Reaction
 RS-SR + R′-SH ↹ R′-S-S-R + RSH
(disulfide isomerase) Coupled with protein-folding
process
 Features of
post-translational
modifications
Review of Translation:
Purposes of Post-translational Events
and Modifications
A. Quality Control: Chaperones, Glycosylation

B. Degradation of misfolded proteins: Ubiquitination, ERAD

C. Proper protein function: Glycosylation, Phosphorylation,

Ubiquitination

D. Target protein to proper locations: Acylation, GPI anchors

Quality Control in the Cytoplasm:
A. Anfinsen's dogma:
 All information needed for folding contained in the amino acid

sequence:
 Leads to the concept of spontaneous protein folding.

B. Problems with Anfinsen's dogma (and the notion of

spontaneous folding):
 Features of cellular environments cause misfolding &

aggregation.
1. Some proteins take a very long time to fold
spontaneously.
2. Some protein domains are prone to misfolding and
aggregation.
Quality Control in the Cytoplasm:
B. Problems with Anfinsen's dogma, cont. Protein folding in vivo
Folding in the cell differs from refolding of a
denatured protein in vitro due to: final folded structure
nascent chain
Vectorial nature of protein synthesis in vivo.

Exposure of hydrophobic regions during synthesis.

PRODUCTIVE PATHWAY
Translation happens more slowly than folding,
requiring a “delay” mechanism to allow
translation to "catch up".
aggregation due to exposure of
Highly crowded cytoplasm: 300 mg/ml prot. hydrophobic regions

Folding in vitro is inefficient (20 - 30%); in the cell,

efficiency close to 100%.

Conditions of stress found in vivo exacerbate

misfolding and aggregation.

DEAD-END PATHWAY
Quality Control in the Cytoplasm:

C. Molecular Chaperones:
Proteins that mediate correct fate of other polypeptides but are
not part of the final structure
 Fate includes folding, assembly, interaction with other cellular components,
transport, or degradation.

A. History:
 Molecular chaperones initially identified as heat shock proteins, i.e. proteins
upregulated by heat shock and other stresses.
 Heat shock causes protein denaturation with exposure and aggregation of interactive
surfaces.
 Heat shock proteins inhibit aggregation by binding to exposed surfaces during times
of stress but also during normal protein synthesis
 Thus, the stress response is simply an amplification of a normal function that is used
by cells under non-stress conditions.
D. Features of molecular
chaperones:
 Hsp 70 family members:
 70 kD protein monomers.
 Include DnaJ (bacteria); BiP (ER)
 Stabilize polypeptide surfaces in an unfolded state.
 Bind transiently to newly-synthesized proteins: paradoxically,
Hsp 70
efficient folding requires "antifolding".
 Bind permanently to misfolded protein.
 Have affinity for exposed hydrophobic peptides.
 Do NOT bind a specific sequence.
Hsp 70 stabilizes
 Present in bacteria, eukaryotes & all compartments. the nascent chain
 Regulated by ATP hydrolysis.
 Undergo cycles of binding and release
 Act with cofactors (i.e. DnaJ, GrpE, Hip, Hop, Bag1).
D. Features of molecular chaperones:
ii. Chaperonins (GroEL, Hsp 60, TCP-1):
 Facilitate proper folding
 Bind and hydrolyze ATP
 Bind transiently to 10-15% proteins, but 2-3fold more w/stress
 60 kD proteins that form oligomeric, stacked double rings
 Bring non-native substrate protein to central cavity folding
where protected from aggregation with other non-native
proteins
 Cycles of binding and release until the protein is properly
folded

iii. Others: I.e. small heat shock proteins, hsp 90, etc.
iv. Cytosolic chaperone co-
ordination:

Chaperones act in tandem.

Stabilization by Hsp 70
plus cofactors) may be
followed by use of Hsp 60
for proper folding.
3. Quality control in the ER:
A. Translation and translocation of proteins into the ER:
 Proteins that translocate into ER of mammalian cells include secretory proteins, TM proteins, or
residents of a membranous compartment.
 These are targeted to the ER CO-TRANSLATIONALLY by an N-terminal signal sequence that
generally gets cleaved during translocation across the ER membrane.

The Signal Hypothesis

SRP and SRP Receptor
 Translocation of Secretory Protein

Translocation of Single Pass TM Protein

Translocation of Double Pass TM Protein

3. Quality Control in the ER:
B. Features of the ER:
 Proteins need to be unfolded to translocate
 Until signal sequence cleaved, N terminus of protein is constrained "incorrectly”
 ER lumen is topologically equivalent to extracellular space
 High oxidizing potential (unlike cytoplasm which is highly reduced)
 High Ca+2 concentration unlike cytoplasm
 Many sugars present along with machinery for glycosylation
 As in cytoplasm: high protein conc. (100 mg/ml) promotes aggregation
 As in cytoplasm: delay between translation/ translocation vs. folding
 Site of specific post-translational events: signal cleavage, S-S bond formation, N-linked
glycosylation and GPI anchor addition
3. Quality Control in the ER:
C. Specific ER chaperones:

i. HSP 70 family members: BiP/GRP78

 Recognize hydrophobic sequences in nascent chains.
 Undergo successive rounds of ATP-dependent binding and release.
 Essential for translocation of newly-synthesized proteins across the ER lumen and
for retrograde transport into the cytosol

ii. Immunophilins/ FKBP - peptidyl prolyl isomerases.

iii. Thiol-disulfide isomerases - PDI and ERp57

iv. Calnexin and Calreticulin:

 To the ER
 Are lectins (carbohydrate binding proteins)
 Calreticulin - lumenal; Calnexin - integral membrane protein
4. Selective post-translational proteolysis.
 Selective proteolysis is critical for cellular regulation.

 3 steps for proteolysis in the cytoplasm:

i. Identify protein to be degraded
ii. Mark it by ubiquitination
iii Deliver it to the proteasome, a protease complex that
degrades it

A. The Ubiquitin/Proteasome system:

Ubiquitin:
A highly-conserved 76 aa protein present in all eukaryotes.
Covalently attached to e-amino groups in lysine side chains,
Can be a single ubiquitin or multiple branched ubiquitins.

Signal for ubiquitination can be:

1. Programmed via hydrophobic sequence or other motif
2. Acquired by 1) phosphorylation, 2) binding to adaptor
protein, or 3) protein damage due to fragmentation,
oxidation or aging.
5. Glycosylation in the ER
and beyond:
 Role of sugars in the ER: bulky hydrophilic groups that
maintain proteins in solution, affect protein conformation,
and allow lectins to facilitate folding and exert quality
control.

A. N-linked glycosylation - co-translational;

recognizes Asn-x-Ser/Thr on nascent chain
 Catalyzed by oligosaccharyltransferases - integral
membrane proteins with active site in the lumen.
Transfers a preformed "high mannose" 14-residue
sugar(Glc3Man9GlcNAc2) en bloc to asparagine residues
on the acceptor nascent polypeptide chains. Highly
conserved in mammals, plants, fungi.

i. Donor molecule is dolichol-P-P-Glc3Man9GlcNAc2. Dolichol is

a very long lipid carrier.
ii. Subsequent trimming of residues (also called processing) off
core sugar attached to protein occurs in the ER via
glucosidases and mannosidases.
 Bacteria: no N-glycosylation via dolichol
iii.  -Glucosyltransferase recognizes
misfolded glycoproteins and reglycosylates Yeast: have only oligomannose type N-
them. glycans, because they don't have the
ability to add GlcNac in the trans Golgi
iv. Calreticulin and calnexin serve as sensors
by binding to mono-glucosylated proteins, Since bacteria & yeast lack Glc-Nac
facilitating their folding and assembly. transferase enzyme, this enzyme
v. Only glycoproteins that have been demarcates a fundamental evolutionary
boundary between uni- and multicellular
correctly folded (by calnexin and organisms.
calreticulin), get packaged into ER-to-
Golgi transport vesicles.
vi. In the cis Golgi, further processing &
addition of GlcNac's to form branched
structures
vii. Addition of more sugar residues in the
trans-Golgi (I.e. fucose and sialic acid) to
produce the diversity that is seen in mature
glycans.
Simplified view of N-glycosylation
4. monoglucosylated
proteins are bound
and folded bycalnexin
and calreticulin

1. core sugar 2. trimming 3glucosyl

added en bloc of glucose transferase = Glucose
co-translationally residues adds back
to asparagine in ER glucose = Mannose
residues in ER to unfolded
in nascent chains glycoproteins
(from dolichol = GlcNac
donor)
= Galactose
= Sialic Acid

5. in the
Golgi,
6. in the medial and trimming
trans-Golgi of mannose
more residues
N-acetylglucosamines occurs
and fucose are added as
well as galactoses and
sialic acid (terminal
glycosylation) medial-Golgi cis-Golgi
using GlcNac
transferase
Other post-translational modifications:
A. Disulfide bond formation in the ER
 Protein disulfide isomerase (PDI): in the ER: catalyzes oxidation of disulfide bonds in the
cytosol and at the plasma membrane: reduces disulfide bonds

 Other proteins that act like PDI may be even more important in disulfide bond formation

 Requires action of a regenerating molecule (i.e. glutathione); NADPH is the source of redox
equivalents.

Disulfide Bond Formation

S SH

substrate PDI S
SH redox
S regenerator
S
S SH
SH redox
PDI
regenerator
substrate
S SH
SH
Post-translational Modifications that Alter Location:
A. Acylation - Lipid attachments that anchor proteins to the membranes:
Include myristoylation, palmitoylation, prenylation
Involves addition to protein of fatty acids (long hydrocarbon ending in COOH)
Allows proteins to target to the cytoplasmic faces of membrane compartments
i. Myristoylation: addition of C-14 FA myristate to N-terminus in cytoplasm
 Donor is myristoyl CoA
 Occurs co-translationally in the cytoplasm; can occur post-translationally when hidden motif is
revealed by protein cleavage (i.e. pro-apoptotic protein BID)
 Enzyme NMT recognizes consensus sequence at N-terminus often revealed by a
conformational change (myristoyl switch).
 Promotes weak but typically irreversible interaction with cytosolic membrane face
 Myristoylated proteins traffic through the cytoplasm
 Myristoylation necessary but not sufficient for membrane binding
 Second signal needed for membrane binding: myristate plus basic (basic aa’s interact with
acidic phospholipids PS and PI), or myristate plus palmitate

Myristoylation Met Gly

Removal of initiating methionine

Gly

N-myristoyl transferase (NMT)

Addition of myristate to N-terminal
O
C-N-CH2-C Gly
CH3 H
ii. Palmitoylation - addition of a C-16 fatty acid to the thiol side chain of an internal
cysteine residue.
 Promotes a reversible interaction with membrane
 Palmitoylated proteins traffic to membrane via cytoplasm or via secretory pathway
 Enzymes not well understood
 Myristoylated and palmitoylated proteins are enriched in caveolae and rafts

Palmitoylation

Cys

SH2

Cys
CH 3
S C
H
O
iii. Prenylation - addition of prenyl groups (two types) to S in internal cysteine

a. Farnesylation - C15 fatty acid to C terminus by thioester linkage

 Occurs at CAAX sequences: cys, 2 aliphatic residues and C-terminal residue
 After attachment, last 3 residues are removed and new C terminal methylated
 Creates a highly hydrophobic C terminus
b. Geranylgeranylation - similar to above but addition of C-20 to C terminal Cys

Farnesylation
Cys A A X

SH
addition of farnesyl group

Cys A A X

S
proteolysis

Cys

S
methylation

Cys

S -O-CH 3
Post-translational Modifications that Alter
Location:
B. GPI anchors - Glycophosphatidyl inositol (GPI) attached to the C terminus
 Composed of oligosaccharides and inositol phospholipids
 Provides a mechanism for anchoring cell-surface proteins to the membrane as a flexible
leash that allows the entire protein (except for anchor) to be in extracellular space (unlike a
transmembrane protein)
 Added to translocated proteins in ER
 Targets to PM via secretory pathway
 Unlike with N- or O-glycosylation, no more than ONE GPI anchor per protein
 Unlike acylation, targets proteins to outer leaflet of plasma membrane
 Can be cleaved by PI-phospholipase C (PI-PLC)
 Are minor components on mammalian cells but abundant on surfaces of parasitic protozoa
(i.e. trypanosomes and Leishmania) and yeasts
 Concentrated in lipid rafts
Structure of a GPI anchor:

Protein

C=O C-terminus
NH
CH2 ETHANOLAMINE
CH2
N-Acetylgalactosamine P
Mannose

OLIGOSACCHARIDE

NH3 CH2 CH2 P Glucosamine

Inositol head of
Lipid Bilayer PHOSPHATIDYLINOSITOL
B. GPI anchors - GPI Anchor Formation GPI anchored

Functions: cytoplasm protein tethered

to outer leaflet
of PM
ER lumen protein
translation extracellular
space
ER and GPI
 Stronger anchoring to PM than translocation
acylation cytoplasm GPI
cytoplasm

 Some GPI anchors can be PM

cleavage of
replaced with TM anchors and be ER lumen
hydrophobic
C terminal
functional; others cannot ER sequence and extracellular
space
transfer of
 Crosslinking results in signal preformedGPI
moiety
transdcution across bilayer, cytoplasm
GPI
including Ca influx, tyrosine
phosphorylation, cytokine ER lumen
PM cytoplasm
vesicle
fusion
secretion, etc. ER
 Can interact with TM proteins vesicle
capable of intracellular signaling GPI formation
cytoplasm
 Can indirectly modulate activity
of cytosolic signaling molecules =N terminal
signal sequence ER lumen
vesicle
transport
assoc. w/ lipid rafts =C terminal
GPI signal ER
Why protein post-translational
modifications are made
(Biological functions)
A. Regulation (interconvertable
modifications)
 Monocyclic cascade (allosteric effectors)
-P/de-P receptor-associated Tyr-Kinase coupled
cascade

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

 Unidirectional cascade
Proteolytic activation
B. Cross-links
Stabilize or fix certain folded strands.
(Cofactors covalent binding)

 Disulfide bond-cross links (Cys-Cys)

 Isopeptide (N-(γ-Gln)-Lys)

 Transamidation (Gln→Lys or Ornithine-α-NH2)

(Transglutaminases)
 Blood clotting factor VIII – coagulation
 Tissue Transglutaminase reaction
 Cell proliferation, aging, endocytosis, secretion,
differentiation, apotosis, programmed cell death
 C. Covalent Cofactors
 Biotinyl lysine (Carboxylase, transcarboxylases)

 Cys-bound linear tetrapyrrole (phycobiliproteins

light-harvesting system of photosynthetic
microorganisms)

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

 FMN-linked Cys

 Heme-covalent bound (Cyt. C)

D. Membrane Anchors
 α-NH2 myristoyl

 Cys- fattyacyl thioether

 Ser- palmitate or other fatty acids esters

Thr-

 C-terminal glycophospholipids

 C-terminal Cys – prenylated group

Farnesyl C15, Geranylgeranyl C20
E. Protein Turnover (Protein Degradation)

 Spontaneous Oxidation: Cys, His, Tyr,

Met

 Ubiquitination: Lys
F. Others

 Iodination: Tyr, (Thyroid hormones)

 Sulfation or methylation; Tyr / secreted

proteins
Examples from Pathobiology:
A. ERAD discovered through study of CMV US11 (Wiertz et al., Cell 84: 769, 1996).
1. MHC class I, a TM protein, binds viral peptides produced in cells and presents them at the cell surface to
cytotoxic T cells.
2. CMV evades the immune system by targeting MHC class I for destruction soon after it is synthesized and
translocated into the ER. How does it do this?
3. CMV US11 protein expressed alone causes MHC class I destruction.
4. US 11 effect is sensitive to proteasome inhibitors and involves MHC class I localization to cytoplasm,
implying movemnt of US 11 out of ER into cytoplasm for degradation.
5. Only forward movement thru translocon was thought to occur; this paper by Ploegh’s group studying a CMV
protein raised the possibility of retrograde movement through translocon.

6. Subsequently, retrograde movement thru ERAD:

translocon for degradation (ERAD) was
shown to be a common in non-infected cells.
7. Note that MHC class I needs to be poly-
ubiquitinated for retrograde transport to
occur, implying a role for ubiqutination in
retrolocation, not just in targeting for
degradation.
8. Additional studies reveal that other pathogens
use this mechanism: I.e. HIV-1 accessory
protein Vpu promotes degradation of CD4 by
ERAD.
B. HIV-1 envelope protein
undergoes many critical post-
translational modifications
1. HIV env consists of gp120 soluble portion bound
non-covalently to TM gp41.
Role is to bind CD4 and chemokine receptors
during HIV-1 entry.
2. Co-translationally translocated into ER as gp160.
3. Has ~30 potential sites for N-linked glycosylation
in ER.
If non-glycosylated: won’t bind CD4.
Some glycosylations are dispensible for proper
folding; others are needed.
4. Forms 10 disulfide bonds in ER (9 are in gp120
portion).
Land, A. and I. Braakman, Biochimie 83: 783 (2001).
5. Trimerization of HIV-1 env in ER
6. Proper folding/trimerization equires BiP,
calnexin, calreticulin, and PDI.
7. In Golgi: protease-mediated cleavage of gp160 to
gp120 and gp41.