Proteasome inhibition in live cells:
• Up to 80% of cell proteins are processed by
• Proteasome inhibition in mammalian cells
induces apoptosis and sensitizes cells to pro-
• Rapidly dividing cells are more sensitive than
slowly dividing cells
• MALIGNANT CELL LINES ARE MUCH MORE
The Ubiquitin-Proteasome System
• The proteasome is the main nonlysosomal
endoprotease enzyme complex present in the
cytoplasm and nucleus of all eukaryotic cells.
• It plays a critical role in the degradation of most
short-lived intracellular proteins that control cellular
events such as cell cycle, transcription, DNA repair,
cell death, signal transduction, metabolism,
morphogenesis, differentiation, antigen
presentation and neuronal function.
• The proteasome is also responsible for protein
quality control by eliminating damaged and
• The proteasome is a large hollow and cylindrical 26S enzymatic complex of
at least 66 proteins
• It is composed of the catalytic 20S core and two 19S or 11S regulatory
units at either ends.
• The catalytic 20S core is organized into a stack of four seven-subunit rings,
with the top and bottom rings formed by seven polypeptides, termed the
α-subunits, and the two inner rings of seven β-subunits.
• Poly-ubiquitination drives the interaction
between the 19S (11S) and 20S particles.
It requires the activity of three enzymes:
1. ubiquitin-activating enzyme (E1),
2. ubiquitin-conjugating enzyme (E2)
3. ubiquitin-protein ligase (E3)
• Polyubiquitination each Ub is added
sequentially to the growing chain of target
• 19S regulatory particle has an affinity for and
recognizes these UBL domains.
• The substrate protein is unfolded by hydrolases
so that it may enter the narrow gate of the 20S
particle and then be degraded.
• The 19S regulatory particle is divided into 2
subcomplexes called the BASE and the LID.
• The base consists of 6 AAA+ ATPases & 3 non-
ATPase polypeptide chains.
• The lid includes at least 9 non-ATPase polypeptide
chains that help remove ubiquitin from the
• The lid and base connection is stabilized by the
• The base of hexameric ring of 6 ATPases in the 19S
regulatory particle facilitates the opening of the 20S
gate and is responsible forsubstrate recognition,
deubiquitination, unfolding & translocation into the
• Proteolytic chamber (20S core) - 3 types of catalytic
• chymotrypsin-like β5
• trypsin-like β2
• caspase-like β1
In immune cells the β1, β2, and β5 subunits, which
are constitutively expressed, are replaced by β1i,
β2i, β5i induced subunits to compose the
• Immunoproteasome has increased chymotrypsin-
like and trypsin like activities which assists in
Antigen Processing .
• Instead of the 19S regulatory particle, the
immunoproteasome utilizes the 11S regulatory
Peptide aldehydes (MG132) first
proteasome inhibitors act against serine
and cysteine proteases.
• Peptide aldehyde inhibitors are rapidly
oxidized into inactive acids in cells and
transported out of the cell by the multidrug
resistance (MDR) carrier system
Peptide boronates- Bortezomib
• Peptide boronates bind with the hydroxyl group of the N-
terminal threonine residue in the proteasome by a non-
• The boron atom can receive the oxygen lone pair of the N-
terminal threonine residue stable tetrahedral
• A dipeptide that contains a boronic acid instead of a
carboxylic acid at the C-terminus.
• Bortezomib’s boronic acid reversibly binds to the
chymotrypsin-like β5 subunit of the catalytic chamber of
the 20S particle and inhibits proteasome function
• Bortezomib -> bind to β 5 (20S) disrupts i/c
signaling cascade l/t apoptosis
• NFkB (cytosol) bound to IkB
ubiquitinated degrade proteosomes.
• Under stress IkB gets ubiquitinated
degrade release NFkB enter nucleus
increase transcription of cell survival gene
(cell adhession pr, E selectin , ICAM 1, VCAM
1), Proliferative Pr (cyclin D1), anti apoptotic
molecule (CIAPs, BCL2)
• NFkB expressed in Tx cell help Tx cell to
survive hypoxia, chemotherapy
• Bortezumib also disrupts UPS degradation
of P21,P27, P53 / initiator of apoptosis/ other
key regulator unfolding increase +
• It also sensitize to other cytotox(alk agents/
• Dose 1.3 mg/m2 IV bolus
• Day 1, 4, 8 & 21 (21 day cycle) (10 day gap/
• T1/2 – 5.5hr
Resistance to bortezomib
• Either inherit or acquire mechanisms
1. Mutated or overexpressed (β5)
2. Increased Levels of downstream effectors
(chaperone pr- BIP)
3. Heat shock proteins (resistance to apoptosis )
overexpressing HSP27, 70, and 90 &T cell factor .
4. Constitutive NF-ĸB activity
5. Failing to accumulate pro-apoptotic proteins
6. Increase the levels of anti-apoptotic proteins,
induce autophagy, and increase the levels of anti-
Peptide epoxyketone inhibitors
• α,β-epoxyketone moiety adduct with the N-
terminal threonine residue inactivates
• Carfilzomib is an irreversible inhibitor of the
chymotrypsin-like subunit of the proteasome and
• Undergoing phase II and III trials.
• Carfilzomib is used to treat recurrent multiple
myeloma, non-Hodgkin’s lymphoma and few solid
• Irreversible inhibitor of the chymotrypsin-like,
caspase-like, and trypsin-like activities of the
• In phase Ib
• It is used to treat recurrent multiple myeloma,
solid tumors, lymphomas, and leukemias