Lecture

Fall 2019
BIO702
Gene Regulation and Expression (GRE)
By
M. Jawad Khan, PhD

Sources: Schubert et al. (2000) Rapid degradation of a large fraction of newly synthesized proteins by proteasomes.
Nature 404, 770-774.
www.sfu.ca/leroux/class_L18
 Protein Degradation
Turnover of protein is NOT constant

Half lives of proteins vary from minutes to infinity

“Normal” proteins – 100-200 hrs

Short-lived proteins
Regulatory proteins
Enzymes that catalyze committed steps
Transcription Factors

Long-lived proteins
Special cases (Dentin, Crystallins)
 Protein Degradation
§  Ubiquitin/Proteasome Pathway
80-90%
Most intracellular proteins

•  Lysosomal processes
10-20%
Extracellular proteins
Some intracellular proteins
Two Sites for Protein Degradation

Proteasomes

Large (26S) multiprotein complex (28 subunits)

Degrades ubiquitinated proteins

Lysosomes

Basal degradation – non-selective

Degradation under starvation –
selective for “KFERQ” proteins
The Ubiquitin/Proteasome

PATHWAY
 UBIQUITIN
Ø  Small peptide that is a “TAG”
Ø  76 amino acids
Ø  C-terminal glycine - isopeptide bond with
the amino group of lysine residues on the
substrate
Ø Attached as monoubiquitin or polyubiquitin
chains
Ø Three genes in humans:
•  Two are stress genes (B and C)
G
•  One, UbA as a fusion protein

K
Ubiquitin/Proteasome Pathway

Degradation by the
Ubiquitination 26S PROTEASOME
The Ubiquitin/Proteasome Pathway

Four Main Steps:

•  UBIQUITINATION
•  RECOGNITION
•  DEGRADATION
•  DEUBIQUITINATION
UBIQUITINATED PROTEINS
UBIQUITIN CHAINS
 UBIQUITIN CHAINS
6 11 27 29 33
MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQ
QRLIFAG KQLEDGRTLADYNIQKESTLHLVLRLRGG
48 63
 Functions of Ubiquitination
• Mono-ubiquitination
Ø  Receptor internalization
Ø  Endocytosis – lysosome
Ø  Transcription regulation

•  Poly-ubiquitination
Ø  Targets proteins from Cytoplasm, Nuclear &
ER for degradation by the PROTEASOME
Ø  DNA repair
 Ubiquitination of proteins is a FOUR-step
process
Ø First, Ubiquitin is activated by forming
AMP
a link to “enzyme 1” (E1).

Ø Then, ubiquitin is transferred to one

of several types of “enzyme 2” (E2).

Ø Then, “enzyme 3” (E3) catalizes the

transfer of ubiquitin from E2 to a Lys
amino group of the “condemned”
protein.

Ø Lastly, molecules of Ubiquitin are

commonly conjugated to the protein to
be degraded by E3s & E4s
RECOGNITION
DEGRADATION SIGNALS
substrates
 DEGRADATION
PROTEASOME COMPONENTS
20S 19S
Proteasome Particle

ATP

26S
Proteasome
The 26S proteasome
 Ubiquitinated proteins are
degraded by the proteasome
Ø Ubiquitinated proteins are degraded in the
cytoplasm and nucleus by the proteasome.

Ø Proteasomal protein degradation consumes

ATP.

Ø The proteasome degrades the proteins to ~8

amino-acid peptides.

Ø Access of proteins into the proteasome is

tightly regulated.

Ø The peptides resulting from the proteasome

activity diffuse out of the proteasome freely.
 DEUBIQUITINATION

De-ubiquitinating
 Ubiquitin – like proteins
“UBP”

Small
Ubiquitin-like
Modifier
Ubiquitin – like modifiers
 Ubiquitin-fold Structures
v  Ubiquitin, Nedd8, UBX and ThiS all have ubiquitin folds
(i.e., similar structures)
v  ThiS is the most divergent, with only 14% sequence identity
with ubiquitin; in comparison, Nedd8 is >50% identical
v  All of the sequences contain C-terminal Gly-Gly residues
that is used for conjugation; this Gly-Gly terminus is perfectly
conserved

ubiquitin Nedd8 UBX ThiS

 ThiS
v  ThiS is a sulfur ‘carrier’ protein that plays a central role in
thiamin biosynthesis in E. coli
v  During thiamin biosynthesis, sulfur from Cysteine is
transferred to thiazole, which is then incorporated into thiamin

O O
ThiF ThiF
ThiS ThiS ThiS
ATP Cys
AMP SH
ThiF, ThiH,
Thil, ThiG
S OP
S OP
+
N
N N thiazole
thiamin
N reaction scheme
NH2 for the biosynthesis
of thiamin
 Molybdopterin synthase
v  Molybdopterin (MPT) synthase is an evolutionarily-conserved enzyme
that is present in bacteria, archaea and eukaryotes; it is a dimeric protein
that consists of MoaD and MoaE, and together with MoeB catalyzes the
formation of Molypdopterin
v  Molypdopterin plays a role in sulfite detoxification and the metabolism
of xenobiotics
v  Defects in molydopterin biosynthesis results in human disease
OH
O
O
P
OH
O
O MoaE
precursor z
MPT synthase
(MoaD, MoaE)
and MoeB
SH
SH
MoaD
ubiquitin (Ub-like fold)
OPO3- for comparison
O
molybdopterin