University of Lucknow

1. “
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UNIVERSITY OF LUCKNOW
Department of Biochemistry
Ubiquitin-proteasome protein
degradation pathway
Presented by :
SAMIDHA
M.Sc. Biochemistry (Sem.-II)

2. Introduction-
 The levels of proteins within the cells are determined not
only by rates of synthesis but also by rates of degradation.
 Many rapidly degraded proteins function as regulatory
molecules , such as Transcription Factors.
 The rapid turnover of these proteins is necessary to allow
their levels to change quickly in response to external
stimuli.

3.  Others proteins are rapidly degraded in response to
specific signals , providing other mechanism for the
regulation of intracellular enzyme activity.
 In addition faulty or damaged proteins are recognized and
rapidly degraded within cells , there by eliminating the
consequences of mistake during protein synthesis.
 In Eukaryotic Cells two major pathways –
I. Ubiquitin-Proteasome Pathway
II. Lysosomal Proteolysis Mediate Protein Degradation

4. PROTEASOME -
Proteasomes are protein complexes which degrade
unneeded or damaged proteins by proteolysis , a chemical
reaction that break peptide bond . Enzymes that help such
reaction are called proteases.
Proteins are tagged for degradation with a small protein
called ubiquitin.
The tagged reaction is catalyzed by enzyme called
ubiquitin ligases.

5. STRUCTURE-
 In structure the proteasome is cylindrical complex containing
a ‘core’ of four stacked ring forming a central pore.
 Each ring is composed of seven individual proteins.
 The inner two rings are made of seven β subunits that contain
three to seven protease active sites.
 These sites are located on the interior surface of the rings so
that the target protein must enter the

6. Central pore before it degraded.
The outer two rings each contain seven α subunits whose
function is to maintain gate through which protein enter the
barrel.
Cartoon representation of a proteasome. Its active sites are sheltered inside the
tube (blue). The caps (red; in this case, 11S regulatory particles) on the ends
regulate entry into the destruction chamber, where the protein is degraded
Top view of the proteasome above.

7. Ubiquitin Protein -
 A small regulatory protein of molecular mass 8.6 kDa.
 Exists ubiquitously in most tissues of Eukaryotic
Organism
 In addition to being small ubiquitin is made up to 76
amino-acid peptide that is highly conserved in all
eukaryotes ( yeasts , animals and plants ).

8. X-Ray structure of Human Ubiquitin
Cartoon representation of ubiquitin protein, highlighting the secondary structure. α-helices are coloured in blue and
β-strands in green. The sidechains of the 7 lysine residues are indicated by orange sticks. The two best-
characterised attachment points for further ubiquitin molecules in polyubiquitin chain formation (lysines 48 &
63) are labelled

9. Ubiquitylation :
 The addition of ubiquitin to substrate protein is called
Ubiquitylation or Ubiquitination or Ubiquitinylation.
 Ubiquitylation affects proteins in many ways :
 Marking them for degradation via the proteasome
 Altering cellular location
 Can also promote or prevent protein interaction

10.  Ubiquitylation involves three main step :
Activation
Conjugation
Ligation
 The result of this sequential cascade is to bind ubiquitin to
lysine residue on the protein substrate via an isopeptide
bond, cysteine residues through thio-ester bond , or the
amino group of proteins N-terminus via a peptide bond.

11. UBIQUITIN -ACTIVATING ENZYME (E1)
Carboxyl end of ubiquitin is linked to the E1
enzyme through thio-ester bond
UBIQUITIN-CONJUGATING ENZYME(E)
Activated ubiquitin is transferred to cysteine
residues of (E2)
UBIQUITIN-LIGASE(E3)
Transfers the ubiquitin from E2 to target protein
ATP dependent
process
Enzymes

12. Steps of Ubiquitylation :
1. Activation :
o The activation step is catalyzed by ubiquitin enzyme
E1 upon ATP hydrolysis .
o During the activation the ubiquitin molecule
carboxyl glycine residue is linked to the E2 enzyme
cysteine residue via the thio-ester bond . ( cysteine
residue being the active site of the enzyme )

13. 2. Conjugation :
o The step is catalyzed by Ubiquition Conjugating enzyme
E2
o The E2 enzyme catalyzed the transfer of ubiquitin from
E1 to the reactive cysteine site of E2 via a thio-
esterification reaction
o The E2 enzyme binds both the activated ubiquitin and
E1 enzyme

14. 3. Ligation :
o Catalyzed by Ubiquitin Ligase E3
o This enzyme shuttles the ubiquitin to the target protein
thus the substrate recognition module of the system
o It is the enzyme that catalyzes the formation of the
Iso-peptide Bond between the Lysine of the target
protein and the C-terminal Glycine residue of
ubiquitin protein.

15.  The substrate recognition module interacts with both
E2 and the substrate for the ubiquitin transfer
 Once the complex has been made E3 detaches
releasing the ubiquitin substrate protein complex and
E2 enzyme
 Ubiquitin chain elongation factor are capable of
adding preformed polyubiquitin chains to the
substrate proteins

16. UBIQUITIN PROTEIN
PROTEIN
COVALENTLY
LINKED TO
LYSINE RESIDUE
PROTEIN DEGRADATION
ISOPEPTIDE BOND
UBIQUITINATION

17. Ubiquitin-proteasome Pathway
PROTEIN DEGRADATION PATHWAY

18. UBIQUITINATION-
The process begins with the attachment of a small
protein called ubiquitin to the target protein that needs
to be degraded.
This attachment is carried out by a series of enzymes
known as E1,E2 and E3.
The E1 enzyme activates ubiquitin ,which then
transferred to the E2 enzyme before being ligated to
he target by the E3 ligase.

19. POLYUBIQUITINATION-
 The type of ubiquitin chain (e.g., K48-linked, K63-linked )
can determine the fate of the target protein.
 In many cases , multiple ubiquitin molecule are attached to
the target protein, forming a polyubiquitin chain .

20. Recognition And Proteasomal Degradation-
The polyubiquitinated protein is recognized by the
proteasome , a large protein complex that acts as
cellular ‘garbage disposal’.
The proteasome unfolds unfolds the target protein and
degrade it into small peptides.

21. DEUBIQUITINATION-
After degradation , the ubiquitin molecules are
removed from the target protein by deubiquitinating
enzymes (DUBs).
These ubiquitin molecules can be recycled and reused
in subsequent ubiquitination reaction.

22. REGULATION AND SPECIFICITY-
The ubiquitin-proteasome pathway is highly regulated
to ensure specificity and proper targeting of proteins
for degradation.
Different E3 ligases recognize specific target proteins
based on sequence motifs, post-translational
modifications , or other signals.

23. THE UBIQUITIN-
PROTEASOME
PATHWAY
CELL CYCLE REGULATION
PROTEIN QUALITY
CONTROL
DNA REPAIR
SIGNAL TRANSDUCTION
IMMUNE RESPONSE
The ubiquitin-proteasome pathway involves

24. References -
 Proteasome – Wikipedia
 https://journals.lww.com/jasn/fulltext/2006/07000/protein_degradation_by_the_ubiq
uitin_proteasome.14.aspx National library of medicine
 https://www.nature.com/articles/s41392-022-00966-4
 The Cell : A Molecular Approach by Geoffrey M. Cooper , Robert E. Hausman 3rd
Edition
 Biochemistry by Donald Voet , Judith G. Voet 4th Edition
 Images : the Cell : A Molecular Approach

25. THANK YOU !!