This document summarizes a seminar on the ubiquitin-proteasome pathway. It provides an introduction to the topic, a brief history of its discovery, and descriptions of key components of the pathway including ubiquitination, the 26S proteasome, proteasome inhibitors, and disorders related to defects in the pathway. The ubiquitin-proteasome pathway plays an important role in regulating intracellular protein levels and defects can lead to diseases such as cancer, neurodegeneration, and genetic disorders.

1. Seminar Topic:
Presented by: Under Guidance of:
Ms. Pooja R J Dr. Prabhakar B T
I Sem I MSc Assistant Professor
Department of Biotechnology Department of Biotechnology
Sahyadri Science College, Sahyadri Science College,
Shimoga. Shimoga.

2. CONTENTS
1. Introduction
2. History
3. The ubiquitination system
4. The ubiquitin proteasome pathway
5. PA 700, the 19s regulator
6. Proteasomes
7. Degradation of ubiquitinated proteins by 26s proteasomes
8. The ubiquitin proteasome pathway disorders
 Inherited disorders
 Acquired disorders
9. Proteasome Inhibitors
10. Conclusion
11. References

3. INTRODUCTION
• All intracellular proteins and many extracellular proteins are continually “turning
over” i.e. they are being hydrolyzed to their constituent amino acids and replaced
by new synthesis.
• Although the continual destruction of cell proteins might seem wasteful, this
process serves several important homeostatic functions.
• Individual proteins in the nucleus and cytosol, as well as in the endoplasmic
reticulum (ER) and mitochondria, are degraded at widely differing rates that vary
from minutes for some regulatory enzymes to days or weeks for proteins such as
actin and myosin in skeletal muscle or months for hemoglobin in the red cell.
• Cells contain multiple proteolytic systems to carry out the degradation process and
complex regulatory mechanisms to ensure that the continual proteolytic processes
are highly selective.

4. • The discovery of protein ubiquitination is a fine example of a “bottom to top”
discovery and its role in ‘big’ biological processes, e.g. transcription, cell cycle,
antigen processing, cellular defense, signaling etc.
• In most overall, the rates of protein synthesis and degradation in each cell must be
balanced precisely because even a small decrease in synthesis or a small
acceleration of degradation, if sustained, can result in a marked loss of mass in the
organism. In all tissues, the majority of intracellular proteins are degraded by the
ubiquitin (Ub)–proteasome pathway (UPP).
• This process is accelerated by the lack of insulin or essential amino acids and in liver
by glucagon. There are other cytosolic proteolytic systems in mammalian cells. The
Ca2+-activated (ATP-independent) proteolytic process involves the cysteine
proteases termed calpains.

5. DISCOVERY

6. HISTORY
Year Scientist Invention
1977
Etlinger and
Goldberg
Development of a rabbit reticulolysate system to
study non-lysosomal and ATP-dependent protein
degradation
1978
Ciechanover
Fractionation of reticulolysate led to the
identification of two fractions, active principle of
fraction (APF)-I and APF-II. The combination of APF-I
and APF-II reconstituted protein degradation
1979
Hershko
APF-II was sub fractionated into two fractions: APF-
IIa and APF-IIb. APF-IIb contained the E1-E3
ubiquitin conjugating enzymes.
1986
Hough APF-II a was later shown to contain proteasomes.
1980 Wilkinson APF-I identified as ubiquitin.

7. Year Scientist Invention
1980 Hershko
High molecular conjugates of ubiquitin and
substrate proteins were formed in the presence of
conjugating enzymes and ATP.
1980 Hershko
Deubiquitinating enzyme activity identified that
was capable of recycling ubiquitin bound to
substrate proteins.
1981 Hershko
The carboxyl terminal glycine of ubiquitin was
found to be activated by the E1 enzyme.
1982
Hershko
The number of ubiquitin-protein conjugates
increased in reticulocytes during the formation of
abnormal proteins, demonstrating a link between
ubiquitination and protein degradation.

8. THE UBIQUITOUS SYSTEM (UPS)
Figure 1. Overall view of the cytosolic protein degradation pathway in mammals.
• The cytosolic protein degradation pathway leading to free amino acids is represented in this schematic diagram. The
proximal steps in this pathway are ATP-dependent and performed by 26S proteasomes whereas the latter steps are executed
by ATP- independent proteases and peptidases.
• In addition, this pathway also generates 8–15 amino acid long peptides that are transported into the endoplasmic reticulum
and bind to MHC class I molecules for perusal by CD8+ T cells.
• Binding of these peptides to MHC class I protects them from further degradation by exopeptidases Although 26S
proteasomes recognize poly-ubiquitin as tedious degradation signals and contribute to selectivity and specificity of the UPS.

9. UBIQUITIN PROTEASOME PATHWAY
Figure 2. The ubiquitin (Ub)-proteasome pathway (UPP)
of protein degradation.
• Ub is conjugated to proteins that are destined for
degradation by an ATP-dependent process that
involves three enzymes.
• A chain of five Ub molecules attached to the protein
substrate is sufficient for the complex to be recognized
by the 26S proteasome.
• In addition to ATP-dependent reactions, Ub is removed
and the protein is linearized and injected into the
central core of the proteasome, where it is digested to
peptides.
• The peptides are degraded to amino acids by
peptidases in the cytoplasm or used in antigen
presentation.

10. Rapid Removal of Proteins
 Unlike most regulatory mechanisms, protein degradation is inherently irreversible.
 Destruction of a protein can lead to a complete, rapid, and sustained termination of the process
involving the protein as well as a change in cell composition.
 The rapid degradation of specific proteins permits adaptation to new physiologic conditions
 Regulation of Gene Transcription
 Ub conjugation affects transcription by multiple mechanisms.
 Many transcription factors are ubiquitinated and degraded by the proteasome.
 In fact, in many cases, transcriptional activation domains and signals for Ub conjugation di-
erectly overlap.
 Ubiquitination and proteolysis of activators even may stimulate transcriptional activity by
removing “spent” activators and resetting a promoter for further rounds of transcription.

11. Figure 2. The protein ubiquitination pathway. Ubiquitin (Ub) is activated by E1 and transferred to the E2 enzyme and
is, finally, conjugated to substrate proteins with a specific E3 ligase. Further polyubiquitination is required to target
proteins for degradation.

12. • The Lys-48, of ubiquitin is located on the surface of a Type III reverse turn, providing its ε-amino group for an
easy access to form an additional amide isopeptide bond to generate multi ubiquitin chains.
• In the first step of the ubiquitin conjugation cascade, the carboxyl group of Gly-76 of ubiquitin is activated by
ubiquitin-activating enzyme (E1). This step involves the hydrolysis of ATP to PPi to generate an ubiquitinyl
adenylate intermediate bound to an E1 enzyme.
• Subsequently, an active site Cys residue of E1 covalently links to ubiquitin via a high-energy thioester linkage,
with the concomitant release of AMP. Following activation, activated ubiquitin is then transferred by
transacylation reaction to a thiol group of an active site Cys residue of E2 (ubiquitin carrier protein or
ubiquitin-conjugating enzyme).

13. • Finally, E2 shuttles ubiquitin either directly to a protein substrate by itself or in cooperation with
ubiquitin-protein ligase (E3), to form an amide isopeptide bond between the carboxyl group of Gly-76
of ubiquitin and an ε-amino group of the protein substrate’s internal Lys residue.
• The last step occurs by first transferring ubiquitin from E2 to E3, which accepts ubiquitin in a similar
thiol linkage and then to the protein substrate.
• In some cases, however, covalent linkage between E3 and ubiquitin is not observed and it appears that
ubiquitin is directly transferred from E2 to the protein substrate in a ternary E2-E3-substrate complex.
• Once the protein substrate is mono ubiquitinated, a polyubiquitin chain is formed through the same
ubiquitination conjugation cascade,

14. PA700, THE 19S REGULATOR
• Figure 4. Formation of 26S proteasome.
• These are formed by the combination of
catalytic 20S proteasome with PA700, also
known as 19S regulators, in an ATP-dependent
manner.

15. • PA700 is a large complex comprising several subunits, which impart the complex with diverse activities:
ATPase, ubiquitin-binding, deubiquitinating, reverse chaperone.
• PA700 from S. cerevisiae harbors at least 17 subunits, regulatory particle non-ATPase and regulatory
particletripleA-ATPase.
• PA700 can be dissociated into two sub-complexes, base consisting of nine subunits and lid consisting of
eight subunits. The base harbors six essentialia’s and three nonATPase subunits, including the
polyubiquitin-interacting protein S5a.
• These ATPases belong to the ATPases associated with various cellular activities (AAA)-ATPase family. By
utilizing its ATPase activity, the base complex acts as a reverse chaperone to unfold target proteins and also
facilitates opening up the narrow pore of 20S by utilizing its ATPase activity.

16. PROTEASOME
Figure 5. Comparison of the subunit composition of
20S proteasomes from different organisms.
• The proteasome of archaebacterium T. acidophilum
contains single α and β proteasome subunits and
the outer rings are composed of identical α
subunits whereas the inner rings are composed of
the identical β subunit.
• On the other hand, yeast proteasomes are
composed of seven different α and β subunits.
• In mammals, three constitutive proteasomal β
subunits, β1, β2and β5, are replaced by β1i, β2i,
and β5i, which are induced in response to
inflammatory signals.

17. MOST CELL PROTEINS ARE DEGRADED
BY THE 26S PROTEASOME
Figure 6. The 26S proteasome structure
• It includes the 20S core that contains the
unique proteolytic sites that break peptide
bonds.
• The 19S caps cleave off the Ub chain and use
ATPases to unfold the protein substrate and
translocate it into the 20S core particle for
degradation into peptides.

18. HUMAN DISEASES DUE TO THE DEFECTS OF THE
UBIQUITIN PROTEASOME PATHWAY
INHERITED DISORDERS
A. Angelman’s Syndrome
 Angelman’s syndrome is a complex neurological
disorder due to various genetic mechanisms that
map to human chromosome 15q11– q13.
Proteasome pathway plays an important role during
brain development and perturbation of this pathway
causes a neuropathological disorder.

19. B. Liddle syndrome
• Liddle et al. recognized this rare hereditary disorder in a family who were unable
to maintain the proper balance of salt and water in the body, resulting in
abnormally high blood pressure.
• Among many different mutations, deletion or mutation of the proline-rich (PPxY)
region leads to constitutive activation of the kidney sodium channel, resulting in
retention of excessive amounts of salt and water.
• The findings suggests a novel mechanism regulating sodium reabsorption, in
which failure of proper ubiquitination of the sodium channel results in increased
retention of the sodium channel at the cell surface and thus excessive
reabsorption of sodium and water leading to hypertension.
• It should be noted, however, that this short-lived sodium channel, after
ubiquitination, is targeted and degraded in the lysosome, but not by the 26S
proteasome machinery.

20. ACQUIRED DISORDERS
 Cell proliferation, apoptosis, angiogenesis and motility, processes
with particular importance for carcinogenesis are regulated by the
ubiquitin-proteasome system (UPS).
A. CERVICAL CANCER
 The most important risk factor for cervical cancer is human
papillomavirus (HPV) infection.
 cervical cancer caused by the high-risk strains. HPV, levels of the
tumor suppressor protein p53 were found to be unusually low due
to the presence of the E6 proteins encoded by the oncogenic HPV.
 This E6-promoted degradation of p53 involves the ubiquitin-
proteasome pathway and represents a major mechanism

21. B. Colorectal cancer
• Colorectal cancer, which arises in the epithelium of
the lumen of the colon and rectum.
• In most colorectal cancers APC (Adenomatous
Polyposis Coli) disabling mutations interfere with
the ability of the proteasome to degrade β-catenin
leading to uninhibited cell proliferation.
• Other key molecules in colorectal carcinogenesis
such as p53, Smad4 and components of the k-ras
pathways are also regulated by the UPS.

22. C. Neurodegenerative disorders
• Isoforms of ubiquitin-1 have been found in lesions
associated with some neurodegenerative disorders,
such as Alzheimer and Parkinson’s disease.
• Abnormally high levels are thought to decrease the
malformation of amyloid precursor protein (APP),
which is a trigger for Alzheimer’s disease. Abnormally
low levels, on the other hand, lead to an increase in
APP malformation.
• Ubiquitin B changes and mutations may also lead to a
lack of C-terminal glycine in some peptides, called
UBB+1, which is thought to accumulate in
neurodegenerative diseases.

23. A. Synthetic Proteasome Inhibitors
 Peptide aldehydes (MG132) are the first proteasome inhibitors act against serine and cysteine
proteases. The aldehyde functional group is readily subject to a nucleophilic attack by hydroxyl groups
and that the proteasome uses the hydroxyl group of the amino terminal threonine as a nucleophile.
 Peptide boronates – Bortezomib bind with the hydroxyl group of the N- terminal threonine residue in
the proteasome by a non- covalent bond.
 Peptide epoxyketone inhibitors Carfilzomib are α,β-epoxyketone moiety and adduct with the N-
terminal threonine residue and inactivates proteahesome function.
 Carfilzomib is used to treat recurrent multiple myeloma, non-Hodgkin’s lymphoma and few solid
tumors.
 Β-lactone-γ-lactam inhibitors Marizomib are Irreversible inhibitor of the chymotrypsin-like, caspase-
like, and trypsin-like activities of the immunoproteasome.
 Marizomib is used to treat recurrent multiple myeloma, solid tumors, lymphomas, and leukemias.
PROTEASOME INHIBITORS

25. • While the synthetic inhibitors mentioned above have been rationally designed,
synthesized, and optimized, nature has also provided selective and potent proteasome
inhibitors.
• Lactacystin is a Streptomyces lactacystinaeus metabolite that was discovered on the
basis of its ability to induce neurite outgrowth in the murine neuroblastoma cell line
Neuro-2a.
• Epoxomicin from an unidentified actinomycete strain No. Q996-17 displays specific
activity against B16 murine melanoma.
• Eponemycin, an anti-angiogenic linear peptide α′,β′-epoxyketone isolated from
Streptomyces hygroscopicus No. P247–271 on the basis of its specific activity against
B16 melanoma.
• Linear peptide α′,β′-epoxyketone natural products have been isolated directly on the
basis of proteasome inhibition screening from microbial metabolites.
Examples include TMC-86A and B from Streptomyces sp. TC 1084,255 TMC-89A and B
from Streptomyces sp. TC 1087256 and TMC-96 from Saccharothrix sp.
B. Natural Product Proteasome Inhibitors

26. CONCLUSION
• Ubiquitin-proteasome pathway is involved in so many critical cellular processes.
• Proteasome inhibitors are of great interest as potential therapeutics. Indeed, a boronate inhibitor
and a lactacystin inhibitor are currently in clinical trials for cancer and for stroke-associated
ischemia reperfusion injury, respectively.
• In order to improve the safety of proteasome inhibitor drugs and enable more specific inhibition
of biological processes, however, attempts to target earlier steps of ubiquitin-proteasome pathway
are desirable.
• Although such inhibitors are not yet available, the growing amount of information about cellular
substrates and components of ubiquitin conjugation cascade may aid the design of potential E2 or
E3 inhibitors.

27. 1. Ayhyuk Myung, Kyung Bo Kim, Craig M. “The ubiquitous-
proteasome pathway” J biol chem. 373:81-83,1998.
2. Dipankar Nandi, Dilip chandu, and Anujith Kumar, “The ubiquitin-
proteasome system” Journal of Biosciences.31 137-155,2006.
3. Mitch WE, Goldberg AL: the role of the ubiquitin-proteasome
system. N Engl J Med 335:1897-1905, 1996.
REFERENCES

29. THANK YOU