Lecture 15: Post-translational modification of proteins Flashcards
1
Q
How is protein diversity created in humans?
A
- ~21,000 genes in humans
- Greater than 21,000 proteins
- Mechanisms for protein diversity
include:
a. Alternative splicing of pre-mRNA
b. Post-translational modification;
2 types
i. Permanent
ii. Temporary
2
Q
What are different post-translational modifications?
A
- Modification of “non-standard” AAs
a. gamma-carboxyglutamic acid
b. hydroxy-proline - Cross-linking of AAs
a. S=S formation between Cys
sidechains (intracellular)
b. Transglutamination (Gln and Lys
residues; extracellular
3. Addition of other functional groups to
AAs
a. Carbohydrates: glycosylation
b. Lipids: e.g.,smyristylation,
farnesylation, GPI anchors
Often used for membrane
attachment
- Reversible modifications
a. Phosphorylation
b. Acetylation
c. Methylation
d. Ubiquitinoylation - Proteolytic processing
3
Q
How many amino acids can be modified?
A
12/20
4
Q
Elaborate on the modification of “non-standard” amino acids
A
- Hydroxylation of Pro and Lys residues
a. “hydroxylysine”
b. “hydroxyproline” - Almost EXCLUSIVELY in collagen,
essential for maturation and secretion - Occur at sequences:
a. X-Pro-Gly
b. X-Lys-Gly - Reactions occur in endoplasmic
reticulum - Hydroxylase enzymes dependent on
vitamin C
a. Effects of Vit C deficiency (scurvy)
due to defects in collagen synthesis
and stability
5
Q
What does the hydroxylation of proline and lysine residues do?
A
- Hydroxyproline most common post-trans
modification in vertebrates - Stabilises collagen triple-helix (allows
H-bonding) - Hydroxylysine allows covalent
crosslinking of chains
6
Q
Explain the non-standard modification of gamma-carboxyglutamic acid (Gla)
A
1. Modification catalysed by "vit K-
dependent carboxylase"
2. Reaction occurs in endoplasmic
reticulum
3. Gla = strong chelator of calcium ions
4. Occurs in some blood clotting proteases
a. Vit K-dependent coagulation factors:
VII, IX, X, and prothrombin)
5. And Bone proteins
a. e.g., MGP (Matrix Gla Protein)
7
Q
Elaborate on glycosylation (addition of functional groups to sidechains)
A
- Carbohydrates covalently attached
- Many attached to cell membranes
- N-linked attachment to Asn
(asparagine) in sequences:
a. Asn-X-Ser
b. Asn-X-Thr - Also O-linked glycosylation to serine
residues
GlcNAc = N-acetyl glucosamine GalNAc = N-acetyl galactosamine
8
Q
What are N-linked oligosaccharides consisted of?
A
- Pentasaccharides:
a. 2x N-acetylglucosamine
b. 3x Mannose sugars (added
preformed)
Can up to 30% more weight to protein!
9
Q
How does glycosylation take place?
A
- In lumen of endoplasmic reticulum and
Golgi complex - Proteins synthesised by ribosomes that
are attached to ER membrane - N-linked glycosylation begins in ER to
continue in Golgi - O-linked glycosylation takes place in
Golgi only
10
Q
What are the functions of glycosylation?
A
- Increase protein solubility
- Increase protease-resistance
- Can change functional properties of
proteins - Important for protein-protein and cell-cell
interactions
a. During inflammation E-selectin on
endothelial cells recognises
glycoproteins on leukocytes - Modifies antigenic properties:
a. ABO blood groups: A + B have one
extra monosaccharide unit
11
Q
Elaborate on lipids in the “addition of other functional groups” to AAs
A
- Types include:
a. Myristoylation: addition of myristic
acid to alpha-amino group of N-
terminal glycine
b. Farnesylation: addition of palmitic
acid and farnesyl lipids to cystine
residues (reversible)
c. GPI anchors
2. Membrane proteins associate with
membrane through hydrophobic
surfaces
3. Soluble proteins don't have these
surfaces
4. "Allows some soluble proteins
associate with membrane by covalent
linkage to lipid molecules"
12
Q
Give an example of where addition of lipid groups to proteins is useful
A
- G-proteins:
a. guanine nucleotide-binding proteins
b. GTPases - e.g., Ras
Signal transduction pathway becomes more efficient by restricting movement to 2 dimensions
- Ras proteins:
a. 3 lipid groups
b. Part of the signal transduction
machinery so needs to be attached
to membranes
c. Can be a target for stopping
transduction, potential therapeutic role
13
Q
How does the addition of lipid groups aid GPI anchors?
A
- GPI = Glycosylphosphatidylinositol
- No ‘specific’ function
- May help proteins selectively associate
with specific cholesterol-enriched
membrane “domains”
14
Q
Elaborate on phosphorylation as a reversible modification
A
- Most common type of covalent
modification - Catalysed by protein kinases
3. Protein kinases constitute one of largest
protein families (>500 in humans)
- Occurs on:
a. Serine/Threonine
b. Tyrosine
c. Abundance: Ser > Thr > Tyr
15
Q
Why is phosphorylation effective for signal transduction?
A
1. Phosphate group has and adds 2
negative charges to a modified
protein changing shape and structure.
(affects electrostatic interactions and
H- bonding)
2. Stable changes
3. Kinetics can be adjusted from less
than a second to hours
3. Acts as molecular switch: altering
substrate binding and catalytic
activity
4. Amplification: one activated kinase
can phosphorylate hundreds of target
proteins in cascade
