Ubiquitin Proteasome System is a complex machinery for protein degradation.

1. UBIQUITIN-PROTEASOME SYSTEM
Presented by:
Subhojit Ghosh
Roll No: N1930070
M.Sc. in Agricultural Biotechnology
Module: ABT-209
Ramakrishna Mission Vivekananda Educational and Research Institute

2. What is Ubiquitin Proteasome System?
The Ubiquitin-Proteasome system (UPS) is a highly regulated
mechanism of intracellular protein degradation and turnover.
Through the Concerned actions of a series of enzymes,
target proteins are covalently linked to the ubiquitin, which are
then recognized by 26S Proteasome and target proteins are
degraded.
UPS plays important roles in a variety of fundamental cellular
process such as regulation of cell cycle progression, division,
development and differentiation, apoptosis, Cell trafficking
etc.

3. Scientists who discovered Ubiquitin-Proteasome system
The Nobel Prize in Chemistry 2004
Aaron Ciechanover Avram Hershko Irwin Rose
Image source: https://www.nobelprize.org/prizes/chemistry/2004/summary/

4. Ubiquitin
➢ Tag that marks proteins for destruction.
➢ The Ubiquitin itself is a protein Consist of
76 amino acids
➢ Molecular mass is about 8.6 kDa
➢ Ubiquitin is highly conserved in eukaryotes:
yeast and human ubiquitin differ at only 3
of 76 residues.
➢ It contains 7 lysine residues and C-terminal
is glycine where the protein is linked.
Image source: RCSB PDB (https://www.rcsb.org/3d-view/1ubi)
Gly

5. Ubiquitin tags proteins for destruction
• The carboxyl-terminal glycine
residue of ubiquitin becomes
covalently attached by
forming to the Ɛ-amino groups
of several lysine residues on a
protein destined to be
degraded.
• The energy for the formation
of these isopeptide bonds
comes from ATP hydrolysis.
Image source: Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto, Lubert
Stryer, Biochemistry, seventh edition

6. Types of Ubiquitination
Monoubiquitination
S
Ub
S
Ub
Ub
Ub
S
Ub
Ub
Ub
Polyubiquitination
Multiubiquitination

7. How Polyubiquitination Occur?
C terminal of second amino acid is linked by
isopeptide bond to the one of the lysine
residue ( Mostly Lys48 and Lys63) first amino
acid. Similarly third amino acid is linked to
Second amino acid.
Branched Polyubiquitination is also occur
Image Source: https://www.hiclipart.com/free-transparent-background-png-clipart-zmbig

8. Enzymatic Cascade of Ubiquitination
➢ E1: Ubiquitin-activating
enzyme
➢ E2: Ubiquitin-conjugating
enzyme
➢ E3: Ubiquitin-protein ligase
Ring family
HECT family
Image source: Jeremy M. Berg, John L. Tymoczko, Gregory J.
Gatto, Lubert Stryer, Biochemistry, seventh edition

9. What determines whether a protein becomes
ubiquitinated?
➢ Degron
➢ PEST Sequence
➢ Signals in hydrophobic core
➢ The half-life of a cytoplasmic protein is
determined to a large extent by its amino-
terminal residue. This dependency is referred
to as the N-terminal rule or the N-terminal
degron.
E3 enzymes are the readers of N-terminal
residues.

10. Proteasome
• If ubiquitin is the mark of death,
the proteasome is the executioner.
A large protease complex called the
proteasome or the 26S proteasome
digests the ubiquitinated proteins.
• Highly dynamic, multi-subunit, ATP
dependent protease.
• Molecular mass is approximately
2.5 MDa
• The 26S proteasome contains
around 34 different subunit.
Image source: https://doi.org/10.1006/jsbi.1998.3958
26S proteasome structure of Drosophila melanogaster
embryos derived from electron micrographs.

11. Structure of 26 S proteasome
The 26S proteasome is a
complex made up of two
subcomplexes: a catalytic core
particle (20S proteasome) and
two terminal 19S regulatory
particles.
Image Source: https://doi.org/10.1016/S1360-1385(03)00014-1

12. 20S Core Proteasome
The 20S unit is constructed from
14 copies each of two
homologous subunits (α1-7β1-
7-β1-7-α1-7 ).
The subunits are arranged in
four rings of seven subunits that
stack to form a structure
resembling a barrel. The outer
two rings of the barrel are made
up of α subunits and the inner
two rings of β subunits. Image source: https://doi.org/10.2183/pjab/85.12

13. 19S regulatory particles.
• The 19S RP comprises
approximately 20 different
subunits that can be subclassified
into two groups: Regulatory
particle of triple-ATPase (Rpt)
subunits and Regulatory particle of
non-ATPase (Rpn) subunits.
• 19S regulatory particles comprises
two subcomplexes: the lid and the
base
Image source: https://doi.org/10.2183/pjab/85.12

14. Functions of Proteasomal Subunits
❑ Capture Polyubiquitinated Proteins-Rpn10,Rpn13
❑ Deubiquitation-Rpn11
❑ Substrate Unfolding and translocation-Rpt1-6
❑ 20S proteasome gate opening Rpt2, Rpt3, Rpt5
❑ 20S proteasome gate-α1-7
❑ Peptidase activity-β1,β2,β5
Image source: https://doi.org/10.2183/pjab/85.12

15. Degradation in proteasome
The catalytic part of each β1,β2 and β5
Subunits are members of N-terminal
nucleophilic hydrolase superfamily all employ
an N-terminal threonine The hydroxyl group
of the threonine residue is converted into a
nucleophile.
Image Source: https://doi.org/10.1016/S1360-1385(03)00014-1

16. Specificity of catalytic units
Β1: caspase-like(activity with preference acidic amino acid like
glutamate)
β2: trypsin-like (activity with preference basic amino acid like arginine or
lysine)
β5:chymotrypsin:-like ( activity preference hydrophobic amino-acid like
tyrosine or phenylalanine)
All cleavage is done at the peptide carbonyl position of those amino acid.

17. Processing via proteasome
• Length of produced polypeptides: 3-23 amino acids
• Average length of Peptides:7-9 amino acids
• Protein is completely degraded before import of next protein
• Peptides produce by proteasome are further degraded by other
proteases and peptidases.

18. Processes regulated by protein degradation
• Gene transcription
• Cell-cycle progression
• Organ formation
• Inflammatory response
• Tumor suppression
• Biomolecule metabolism etc.

19. Role of Ubiquitin-Proteasome system in Plant body
In addition to the role of E3 ligases targeting specific proteins for
degradation UPS components have vital role in various
mechanism in plant body. Such as
• Abiotic stress signalling: Abscisic acid signalling regulation
through transcription factor during salinity and drought stress.
• Defence system: UPS can lead to either enhanced or reduced
immune signalling, depending on the nature of the disruption.
• Various hormonal regulations.
It would develop an improved utilization of resources for the
better growth of plants.

20. Proteasome Inhibitor Drugs
Program cell death
Proapoptotic signal is generated
Huge amount of damage protein is
gathered.
Proteolysis of damage proteins is
stopped
Inhibitors bind to the proteasome
Proteasome inhibitors are important class of drugs for the
treatment multiple myeloma.
Image source: Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto, Lubert Stryer,
Biochemistry, seventh edition

21. Conclusion
Since the ubiquitin-proteasome pathway degrades a wide range of
protein substrates with exquisite specificity, nature has developed an
ingenious way to divide this complex biochemical machinery into two
discrete and successive steps: first, a specific recognition process,
employing the ubiquitin conjugation cascade, and secondly, an
indiscriminate destruction process, mediated by the proteolytic
proteasome core.
UPS plays important role in controlling of many cellular process and it
has a valuable role in defense system of plant and abiotic stress
signaling.
Proteasome inhibition is an alternative way of cancer therapy.

22. Reference
• Vierstra, R. D. (2003). The ubiquitin/26S proteasome pathway, the complex last
chapter in the life of many plant proteins. Trends in plant science, 8(3), 135-142.
• Walz, J., Erdmann, A., Kania, M., Typke, D., Koster, A. J., & Baumeister, W. (1998).
26S proteasome structure revealed by three-dimensional electron microscopy.
Journal of structural biology, 121(1), 19-29.
• Berg, J. M., Tymoczko, J. L., Stryer, L., & Stryer, L. (2002). Biochemistry. New York:
W.H. Freeman, Seventh edition
• Tanaka, K. (2009). The proteasome: overview of structure and functions.
Proceedings of the Japan Academy, Series B, 85(1), 12-36.
• Nandi, D., Tahiliani, P., Kumar, A., & Chandu, D. (2006). The ubiquitin-proteasome
system. Journal of biosciences, 31(1), 137-155.

23. • Kumar, P., & Mina, U.(2016) Fundamentals and Practice. Life Sciences I, Pathfinder
Publication, Chapter1 Biomolecule and Catalysis.
• Copeland, C., & Li, X. (2019). Regulation of Plant Immunity by the Proteasome. In
International review of cell and molecular biology (Vol. 343, pp. 37-63). Academic
Press.
• Voges, D., Zwickl, P., & Baumeister, W. (1999). The 26S proteasome: a molecular
machine designed for controlled proteolysis. Annual review of biochemistry,
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