Ubiquitin-mediated proteolysis involves three enzymes (E1, E2, E3) that tag unwanted proteins with ubiquitin molecules. The tagged proteins are transported to the proteasome, where they are degraded into peptides. Avram Hershko, Aaron Ciechanover, and Irwin Rose received the 2004 Nobel Prize in Chemistry for discovering this pathway of targeted protein degradation in the late 1970s. The proteasome is a large complex with protease activity that breaks down the tagged proteins.

2. ubiquitin PDB 1TBE

3. • What is ubiquitin mediated proteolysis?
• Who discovered?
• Pathway
• Site of intracellular degradation
• Functions
• Involve in the regulation of the cell cycle

4. • Ubiquitin was first isolated from
bovine thymus but later found in
many different tissues and in
many organisms.
• Ubiquitin is a highly conserved
protein
• Ubiquitin is composed of 76 amino
acid
• Attachment site to target protein on
ubiquitin is C-terminus
• Bond is formed to side chain of Lys
of target protein
• Attachment is performed by array of
enzymes (E1, E2, E3, E4).

6. • Proteolysis is the breakdown of proteins into
smaller polypeptides or amino acids.
• In general, this occurs by the hydrolysis of
the peptide bond, and is most commonly
achieved by cellular enzymes called proteases,
but may also occur by intramolecular
digestion, as well as by non-enzymatic
methods such as the action of mineral acids
and heat.

7. Avram Hershko Aaron Ciechanover Irwin Rose
Nobel prize in chemistry, 2004

8. • A process where an enzyme system tags unwanted
proteins with many molecules of the 76-amino acid
residue protein ubiquitin.
• The tagged proteins are then transported to the
proteasome(a large multisubunit protease complex)
where they are degraded into small peptides.

9. Unwanted protein

11. • E1:
– ubiquitin-activating enzyme.
– Activates the carboxy terminus of
ubiquitin by reaction with ATP.
– exists as two isoforms of 110- and 117-kDa,
are found in both the nucleus and cytosol.
– In mammals there is a single E1.
• E2:
– Ubiquitin-conjugating enzymes.
– E2s are a super family of related proteins.
There are eleven E2s in yeast, and 20-30
E2s in mammals.

13. • E3s:
– Ubiquitin-protein ligases.
– E3s play a key role in the ubiquitin pathway,
as they are responsible for the selective
recognition of protein substrates.
• E4:
– catalyzes the efficient polymerization of very
long polyubiquitin chains, it has been
characterized in yeast.

14. Unwanted protein

15. Three enzymes are involved, designated E1, E2 & E3.
 Initially the terminal carboxyl group of ubiquitin is
joined in a thioester bond to a cysteine residue on
Ubiquitin-Activating Enzyme (E1). This is the ATP-
dependent step.
 The ubiquitin is then transferred to a sulfhydryl group
on a Ubiquitin-Conjugating Enzyme (E2).

16. A Ubiquitin-Protein Ligase (E3) then promotes transfer of
ubiquitin from E2 to the e-amino group of a Lys residue of a
protein recognized by that E3, forming an isopeptide bond.
There are many distinct Ubiquitin Ligases with differing
substrate specificity.
• One E3 is responsible for the N-end rule.
• Some are specific for particular proteins.
ubiquitin C S
O
Cys E2 H2N Lys protein to be degraded
ubiquitin C
O
HS Cys E2N Lys protein to be degraded
H
+
E3
+
(Ubiquitin-Protein Ligase)

17. H2N COO
destruction
box
chain of
ubiquitins
Primary structure of a protein
targeted for degradation
More ubiquitins are added to form a chain of ubiquitins.
The terminal carboxyl of each ubiquitin is linked to the
e-amino group of a lysine residue (Lys29 or Lys48) of the
adjacent ubiquitin.
A chain of 4 or more ubiquitins targets proteins for
degradation in proteasomes.

19. H2N COO
destruction
box
chain of
ubiquitins
Primary structure of a protein
targeted for degradation
Some proteins (e.g., mitotic cyclins involved in cell cycle
regulation) have a destruction box sequence recognized
by a domain of the corresponding Ubiquitin Ligase.

20.  Ubiquitin Ligases (E3) mostly consist of two families:
1) HECT (Homologous to the E6-AP Carboxyl Terminus)-
domain containing a conserved Cys
• Some Ubiquitin Ligases have a HECT domain containing
a conserved Cys residue that participates in transfer of
activated ubiquitin from E2 to a target protein.

21. 2) RING finger-domain
Cys & His residues are ligands to two Zn++ ions
stabilizes a molecular scaffold
Some Ubiquitin Ligases contain a RING finger domain in which Cys &
His residues are ligands to 2 Zn++ ions.
A RING (Really Interesting New Gene) finger is not inherently
catalytic. It stabilizes a characteristic globular domain conformation
that serves as a molecular scaffold for residues that interact with E2.

24. • Ubiquitin—mediated degradation of
cytosolic and membrane proteins occurs
in the cytosol and on the cytosolic face of
the ER membranes.

26. The 20S proteasome core complex encloses a cavity with 3
compartments joined by narrow passageways.
Protease activities are associated with 3 of the b subunits, each
having different substrate specificity.
20 S Proteasome
(yeast) closed state
two views PDB 1JD2


b

b



27. 1. One catalytic b-subunit has a chymotrypsin-like
activity with preference for tyrosine or phenylalanine
at the P1 (peptide carbonyl) position.(hydrophobic a.a)
2. One has a trypsin-like activity with preference for
arginine or lysine at the P1 position.(positive charged
a. a)
3. One has a peptidoglutamyl peptidase like activity with
preference for glutamate or other acidic residue at the
P1 position. (negative charged a.a)
Different variants of the 3 catalytic subunits, with
different substrate specificity.

28. The poly-ubiquitinated protein is recognized by the 26S proteasome, unfolded
and degraded. The ubiquitin molecules are recycled.

29. •Length of produced peptides: 3-23 amino acids
•Average length of peptides: 7-9 amino acids
•Peptide composition of given protein stays constant
•Protein is completely degraded before import of next protein
•Peptides produced by proteasome are further degraded by
other proteases and amino peptidases.
• Proteasome and immune system function:
•Peptides of 8-9 amino acids in length are transported to
the cell surface via the ER presented on the cell surface via
MHC class I – molecules

31. • Eukaryotic cells also contain DUBs
(Deubiquitinating enzymes), which are encoded
by the UCH (Ubiquitin Carboxyl-terminal
Hydrolases) and the UBP (UBiquitin-specific
Processing proteases) gene families.
• UCHs are relatively small proteins (< 40-kDa);in
contrast, UBPs are 50-250-kDa proteins and
constitute a large family.
• Genome sequencing projects have identified more
than 90 DUBs .

35. At the end of summer of 1979 in Fox Chase Cancer Center, Philadelphia. Seated left to right:
Avram Hershko, Sandy Goldman, Jessie Warms, Hanna Heller,
Standing left to right: Zelda Rose, Arthur Haas, Aaron Ciechanover, Mary Williamson,
Irwin Rose, Keith Wilkinson and Leonard Cohen (last three people standing on the
right side not identified)

36. • Advanced information on the Nobel Prize in Chemistry,
6 October 2004,kungl.vetenskapsahedemien,the royal
swedish academy of science Information Department,
P.O. Box 50005, SE-104 05 Stockholm, Sweden.
• Biochemistry- Lubert Stryer
• HARPER’S ILLUSTRATED BIOCHEMISTRY (28TH
EDITION) by Robert murray,david A.bender,peter j
kennekky,victor w rodwell,p.antony weil.