1. The Role of Ubiquitin in
Protein Degredation
and Signal
Transduction

2. Consists of 76 amino acids,
8.5 kDa
Found in all eukaryotic
cells (ubiquitously)
Highly Conserved
Used in post-translational
modification
Ubiquitin

3. 26S Proteasome
Abundant in nucleus and
cytoplasm
destroys proteins marked
by Ubiquitin through
Lysine 48-linked
polyubiquitination

4. 26S Proteasome
Consists of central hollow
cylinder (20S)
4 stacked “rings” of 7
proteins each
Capped by regulatory
particles (19S) that
recognize ubiquitin
through ubiquitin binding
domains (UBDs)

5. Core structure

6. Three types of Ubiquitination

7. Monoubiquitination
Adds one ubiquitin molecule to one substrate
protein residue
Required before a poly chain can begin to
form
Membrane Trafficking, Transcription,
Endocytosis

8. Polyubiquitination
Requires one Ub linked to substrate before
chain begins to form.
Chains made by linking Glysine residue of Ub
to a Lysine of a Ub bound to a substrate.
Linking to different position on Ub leads to
different results.

9. Lysine 48-linked polyubiquitination
Linked by 48th amino
acid (Lysine)
Marks proteins for
destruction
Requires at least 4
Ub to be recognized
by proteasome

11. Lysine 63-linked polyubiquitination
Binds to allow
coordination of
endocytic trafficking
Bound to ESCRT-0
to prevent binding
to proteasome

12. Ubiquitination

13. Ub activating enzyme (E1)
E1 binds ATP and Ub.
Transfers Ub to an active site cysteine
residue, releasing AMP
Thioester linkage between C-terminus of Ub
and E1 cysteine sulfhydryl group
One E1 can transfer Ub to several different
E2 enzymes

15. Ub conjugating enzyme (E2)
Ub is transferred from E1 to E2 through a
trans(thio)esterification reaction.
Binding to both the activated Ub and the E1
enzyme before releasing E1.
Each E2 can transfer Ub to a hundred
different E3 enzymes

18. Ub ligase enzyme (E3)
Attaches Ub via
isopeptide bond to
a lysine on target
protein

19. E3 ligases
The most varied of the three enzymes.
Each E3 can attach to many different
substrate proteins.
Different E2, E3 pairings will recognize
different proteins by distinct degradation
signals.

20. Deubiquitinating enzyme (DUB)
Use catalytic diads or triads
of cysteine, histidine, and
asparagine to catalyze
hydrolysis of the
isopeptide bond

22. Deubiquitinating enzyme (DUB)
Around 100 in the human
genome
Some cleave the whole chain,
some only cleave a set
amount of Ubs
DUB USP5 selectively binds
a 4-ubiquitin chain and
severs it.

23. Ubiquitin in Protein Degradation

24. Ubiquitin in Protein Degradation
After a protein is Ubiquitinated, it must be recognized by
the 19S regulatory particle
Ubiquitin Binding Domains exist to interpret signals from
Ubiquitinated substrates. ~20 different UBDs exist to
bind to different specific shapes of Ubiquitin chains and
different monoubiquitinated locations on a protein.

25. Ubiquitin in Protein Degradation
Narrow gate formed by the N-terminus tails of
the alpha ring subunits
Protein must be partially unfolded, at least
their tertiary structure
Must be deubiquitinated first
Order not clearly known, depends of specific
substrate

26. Proteolysis
Threonine-dependant
nucleophilic attack
Central chamber
releases typically 7-9
residue polypeptides.
Sometimes produce
functioning molecules

28. Regulation of Protein Degradation
One means of controlling
Ubiquitination is
regulating the activation
of E3 ligases.

29. Regulation of Protein Degradation
Another way for a protein to avoid degradation
by the proteasome is to mask the residues
that release the degradation signal.
Phosphorylating the area or creating an
unstable N-terminus will let nearby E3’s
know

30. NF-kB
A protein complex that controls transcription
of DNA.
Synthesized as p105 and p100, C-termini
inhibit entry into nucleus.
Ubiquitinated and processed by the
Proteasome into their active forms, p50 and
p52.

31. Circadian Rhythm and Aging
Ubiquitin is responsible for the degradation of
the “master circadian protein.”
Also damaged proteins that arise due to
aging, stress, and oxidative damage.

32. Ubiquitin in Signal Transduction

34. RIP1
Complexes with a polyubiquitin chain
As long as the signaling protein is
ubiquitinated, it acts to prevent cell death.
Once deubuquitinated by the A20 enzyme,
RIP1 is free to drive forward the cell death
process.

35. PCNA
Monoubiquitination activates PCNA to restart DNA synthesis,
but very error prone.
In yeast, lysine 63-linked polyubiquitination leads to an error
free pathway.

36. Epidermal Growth Factor Receptor
Cell-surface receptor that auto-phosphorylates to activate
downstream activation cascade.
Leads to DNA synthesis, cell proliferation, and cell
adhesion. Important for innate immune response
Ubiquitination by Lysine 63-linkages required for
endocytosis and post endocytic sorting
Mutations to EGFR lead quickly to cancer, proper
ubiquitination prevents out of control mutations.

37. Defects in Ubiquitination Pathway
Tumor suppressor proteins like p53 and p27
are stabilized by Ubiquitin
Defects in Ubiquitin system accelerate
degradation of suppressors, increasing risk
of cancer causing mutations

38. Defects in Ubiquitination Pathway
Alzheimer's
Huntington’s
Parkinson’s
Kennedy’s Syndrome
(Spinobulbar
Muscular Dystrophy)

39. Lewy Bodies
Parkinson’s
Displace other cell
components

40. Ubiquitin-like Proteins (UBLs)
Little is known about most of them
Enzyme cascade is almost the same
SUMO- Small Ubiquitin-like Modifier
Attaches in a manner similar to Ubiquitin, only used in
signal transduction.
ISG15- Interferon Stimulated Gene 15
Expressed in response to interferons or viral dsRNA
Used in JAK-STAT signalingpathway

41. Prokaryotes
Prokaryotic Ubiquitin-like Proteins (PUP)
Attach to substrates in the same manner
Only requires 2 enzymes

42. Prokaryotes