Download Ubiquitin and Ub

18-1
Ubiquitin and Ub-like proteins
Ubiquitin and ubiquitin-like proteins
- background
- the ubiquitin fold
- ThiS, molybdopterin synthase
Degradation
- degradation of newly-synthesized proteins
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Ubiquitin
 Schlesinger and Goldstein (1975) “Molecular conservation of the 74 amino
acid sequence of ubiquitin between cattle and man” Nature 255, 42304.
 note: it is actually 76 amino acids in length
 Wolf et al. (1993) “Ubiquitin found in the archaebacterium Thermoplasma
acidophilum” FEBS Lett. 326, 42.
 Sequencing of the first bacteria and archaea (1995/1996): no sign of ubiquitin!
 ubiquitin is the most highly conserved protein in eukaryotes and is not
found in prokaryotes
 how can such a protein arise in eukaryotes only? Is there not an ancestral
ubiquitin-like protein in prokaryotes?
 ubiquitinated proteins are recognized and degraded by the 26S
proteasome in eukaryotes after a complex ubiquitination pathway
- archaea possess a proteasome as well (20S) but no regulatory
particle which recognizes ubiquitin
ubiquitin is always made as a fusion protein:
N
C
GG
Ubiquitin
fusion protein (often a
ribosomal subunit)
- ubiquitin cleaved after GlyGly by enzyme
Ubiquitin-like proteins were first identified and characterized ~5 years ago
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Ubiquitin-fold structures
 ubiquitin, Nedd8, UBX and ThiS all have ubiquitin folds (i.e., similar
structures)
 ThiS is the most divergent, with only 14% sequence identity with ubiquitin; in
comparison, Nedd8 is >50% identical
 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
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ThiS
 ThiS is a sulfur ‘carrier’ protein that plays a central role in thiamin
biosynthesis in E. coli
 during thiamin biosynthesis, sulfur from Cysteine is transferred to thiazole,
which is then incorporated into thiamin
ThiS
ThiF
ATP
O
ThiS
AMP
ThiF
Cys
O
ThiS
SH
ThiF, ThiH,
Thil, ThiG
S
+
N
N
N
OP
S
N
thiamin
NH2
OP
thiazole
reaction scheme
for the biosynthesis
of thiamin
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Molybdopterin synthase
 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
 Molypdopterin plays a role in sulfite detoxification and the metabolism of
xenobiotics
 defects in molydopterin biosynthesis
results in human disease
OH
O
P
MoaE
O
OH
O
precursor z
O
MPT synthase
(MoaD, MoaE)
and MoeB
SH
ubiquitin
SH
for comparison
OPO3O
molybdopterin
MoaD
(Ub-like fold)
Ubiquitin/Ub-like activation pathways
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 all proteins in ubiquitin superfamily are used in a very similar activation pathway,
with the activating enzyme being homologous
(1)
Gly
ubiquitin
C O
Gly
C O
(2)
ThiS
O
Gly
(3)
MoaD
(1)
O
Uba1 (E1)
E2
*ThiF*
Gly
Gly
Gly
Gly
C
C
C S-E2
C SH
E3
C S-E1
O
AMP
Gly
C S-ThiF
O
AMP
*MoeB*
Gly
C S-MoeB
O
O
ATP PPi
O
(2) Thil, cys
AMP E1
O
ATP PPi
Gly
C
O
ATP PPi
C O
O
Gly
Gly
C NH-protein
O
thiamin
O
Gly
(3) X-S Y
C SH
O
molybdopterin
multiUb’d
protein
transports iron
JAB domain: related to de-ubiquitinating isopeptidase
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Degradation of newly-synthesized
proteins
First: one important use of proteasome degradation products
 proteins are degraded down to 810-mer peptides by the proteasome
 the peptides are then imported into
the ER by the peptide transporter
associated with antigen processing
(TAP)
 MHC class I receptors containing
bound peptides are then presented on
the cell surface
DRiPs
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DRiPs, Defective Ribosomal products
 DRiPs represent polypeptides that never attain native structure owing to errors in
translation or post-translational processes necessary for the proper biogenesis of
the proteins
 Schubert et al.* found that upwards of 30% of all newly-synthesized proteins from
various cell types are degraded by the proteasome
 at least some of the DRiPs are ubiquitinated
 a ubiquitinated DRiP is formed from HIV Gag polyprotein, a long-lived viral
protein that serves as a source of antigenic peptides
 presentation of MHC class I molecules require continuing protein translation,
implying that peptides that are presented are derived from newly-made proteins
- does it make sense that the cell should degrade 30% of its proteins before they
have a chance to function?
- might the degradation of a large fraction of DRiPs represent a defence
mechanism against viral intrusions?
*Schubert et al. (2000) Rapid degradation of a large fraction of newly
synthesized proteins by proteasomes. Nature 404, 770-774.