Ubiquitin Proteasome System (UPS)

 

Ubiquitin-Proteasome System

The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway are the two major degradation systems for both native and misfolded proteins in eukaryotic cells. They do not act independently from each other. Defective autophagy results in the accumulation of ubiquitinated proteins, impacting the flux of the UPS, while dysfunction of the UPS can promote a compensatory induction of autophagy. Through protein degradation and the maintenance of protein homeostasis, the UPS regulates many normal cellular processes including signal transduction, cell cycle control, transcription, and apoptosis (see Figure). The regulated proteolysis of bulk and misfolded proteins is strictly controlled by the 26S proteasome complex.


Figure: The Ubiquitin-Proteasome System Inhibition and affected Cellular Processes



The 26S proteasome complex recognizes polyubiquitinated proteins, which were marked for elimination by the E1, E2 and E3 ubiquitinating enzymes (see Figure). Upon recognition, unfolding and transfer of the de-ubiquitinated target protein by the 19S regulatory cap into the interior of the cylindrical 20S proteasome core particle, protein degradation is facilitated by catalytic β-subunits having nucleophilic N-terminal threonine (Thr1) residues. Although eukaryotic 20S proteasomes harbor seven different β-subunits in their two-fold symmetrical α7β7β7α7 stacked complexes, only three β-subunits per β-ring [subunits β1 (caspase-like), β2 (trypsin-like) and β5 (chymotrypsin-like)] are proteolytically active. These three β-subunits are major targets for small molecule proteasome inhibitors. Proteasome inhibition has implications in a number of human diseases such as cancer (e.g. multiple myeloma (MM)), inflammation and ischemic stroke and is an important therapeutic target.

Several components of the UPS have been validated as potential anticancer targets, including 20S proteasomes, 19S proteasome-associated deubiquitinases (DUBs) and ubiquitin ligases (E3s). One of the strategies to improve the current status of cancer treatment is to repurpose old drugs with UPS-inhibitory properties as new anticancer agents.



STANDARD Proteasome Inhibitors - From the Source! 

Product Name PID Product Description
Bortezomib [PS-341] AG-CR1-3602 Inhibits chymotrypsin-like and caspase-like activity (IC50=3-5nM).
Carfilzomib [PR-171] AG-CR1-3669 Inhibits the chymotrypsin-like β5 subunit of the constitutive 20S proteasome (IC50=5.2nM) and the β5i subunit [LMP7] of the 20S immunoproteasome (IC50=14nM).
Delanzomib [CEP-18770] AG-CR1-3673 Inhibits the chymotrypsin-like β5 subunit of the constitutive 20S proteasome (IC50=3.8nM) and the caspase-like β1 subunit (IC50=~70nM).
(-)-Epigallocatechin gallate AG-CN2-0063 Inhibits chymotrypsin-like activity (IC50~200nm).
Epoxomicin AG-CN2-0422 Inhibits predominant chymotrypsin-like activity (IC50=4nM).
Ixazomib [MLN2238] AG-CR1-3670 Inhibits all the three catalytic activities of the constitutive 20S proteasome: chymotrypsin-like β5 subunit (IC50=3.4nM), trypsin-like β2 subunit (IC50=3.5μM) and the caspase-like β1 subunit (IC50=0.03μM).
Ixazomib citrate [MLN9708] AG-CR1-3671 Inhibits all the three catalytic activities of the constitutive 20S proteasome: chymotrypsin-like β5 subunit (IC50=3.4nM), trypsin-like β2 subunit (IC50=3.5μM) and the caspase-like β1 subunit (IC50=0.03μM).
clasto-Lactacystin β-lactone AG-CN2-0442 Chymotrypsin-like, trypsin-like and caspase-like activity inhibitor (IC50~1μM).
Lactacystin AG-CN2-0104 Chymotrypsin-like, trypsin-like and caspase-like activity inhibitor (IC50=4.8μM).
ONX 0914 AG-CR1-3674 Inhibits the β5i subunit [LMP7] of the 20S immunoproteasome (IC50=73nM) with minimal cross-reactivity to the chymotrypsin-like β5 subunit of the constitutive 20S proteasome (IC50=1.04μM).
Oprozomib [ONX 0912] AG-CR1-3672 Inhibits the chymotrypsin-like β5 subunit of the constitutive 20S proteasome (IC50=36nM) and the β5i subunit [LMP7] of the 20S immunoproteasome (IC50=82nM).
PI-1840 [Proteasome Inhibitor] AG-CR1-3675 Inhibits the chymotrypsin-like β5-subunit of the constitutive 20S proteasome (IC50=27nM), with minimal trypsin-like (β2) and caspase-like (β1) activity (IC50= >100μM, for both).
Piperlongumine AG-CN2-0024 Inhibits the β5i subunit (LMP7) (IC50=15μM) with minimal inhibition of the human constitutive 20S proteasome.
Salinosporamide A [Marizomib] AG-CN2-0444 Inhibits all the three catalytic activities of the constitutive 20S proteasome: chymotrypsin-like (IC50=3.5nm); trypsin-like (IC50=28nm); caspase-like (IC50=430nm).
VR23 [Proteasome Inhibitor] AG-CR1-3676 Inhibits all the three catalytic activities of the constitutive 20S proteasome: chymotrypsin-like (IC50=50-100nm); trypsin-like (IC50=1nm); caspase-like (IC50=3μm).
Z-Leu-Leu-Phe-CHO [MG-110] AG-CP3-0021 Chymotrypsin-like activity inhibitor.
Z-Leu-Leu-Nva-CHO [MG-115] AG-CP3-0015 Chymotrypsin-like activity inhibitor.
Z-Leu-Leu-Leu-CHO [MG-132] AG-CP3-0011 Chymotrypsin-like and caspase-like activity inhibitor (IC50~1μM).
Z-Leu-Leu-Leu-B(OH)2 [MG-262] AG-CP3-0024 Chymotrypsin-like and caspase-like activity inhibitor (IC50~150nM).

* Note: All IC50 values (where indicated) are from literature and might vary based on the experimental setup.




Other Proteasome Inhibitors / Modulators 

Product Name PID Product Description
Apigenin CDX-A0438 Inhibits chymotrypsin-like and trypsin-like proteasome catalytic activity.
Betulinic acid (>99%) AG-CN2-0415 Chymotrypsin-like activity activator at low micromolar concentration.
Betulinic acid (>97%) AG-CN2-0417 Chymotrypsin-like activity activator at low micromolar concentration.
Celastrol AG-CN2-0460 Inhibits 20S proteasome chymotrypsin-like activity.
Curcumin (high purity) AG-CN2-0059 Inhibits all three catalytic activities (IC50~10μM). Inhibits DUB activity.
Kendomycin BVT-0001 Inhibits 20S proteasome chymotrypsin-like activity.
Luteolin AG-CN2-0098 Inhibits chymotrypsin-like and trypsin-like proteasome catalytic activity.
Nelfinavir . mesylate AG-CR1-3726 Pan-proteasome inhibition in AMO-1 and U266 myeloma cells; 60 % inhibition of the chymotrypsin-like activity of 26S proteasome at 5μM.
Quercetin . dihydrate AG-CN2-0409 Inhibits all three catalytic activities (IC50~15μM).
Ritonavir AG-CR1-3683 Inhibits 20S proteasome chymotrypsin-like activity.
Saquinavir . mesylate AG-CR1-3727 Inhibits chymotrypsin-like and caspase-like activity of the 26S proteasome and purified 20S proteasome.
Shikonin AG-CN2-0487 Proteasome inhibitor.
Terrein BVT-0193 Inhibits chymotrypsin- and trypsin-like activity (IC50~0.3mM).
Withaferin A AG-CN2-0490 Inhibits 20S proteasome β5 subunit chymotrypsin-like activity.


Fluorescent Substrates for Ubiquitin-Proteasome Activity Measurement 

Product Name PID Product Description
Ac-Ala-Asn-Trp-AMC AG-CP3-0037 Fluorogenic substrate for specifically measuring chymotrypsin-like activity of the 20S immunoproteasome.
Ac-Arg-Leu-Arg-AMC AG-CP3-0013 Fluorogenic substrate for measuring the trypsin-like peptidase activity of the 20S proteasome.
Ac-Pro-Ala-Leu-AMC AG-CP3-0036 Fluorogenic substrate for specifically measuring caspase-like activity of the 20S immunoproteasome.
Ac-Trp-Leu-Ala-AMC AG-CP3-0035 Fluorogenic substrate for measuring the chymotrypsin-like peptidase activity of the 20S proteasome, calpains and other chymotrypsin-like proteases.
Boc-Leu-Arg-Arg-AMC AG-CP3-0014 Fluorogenic substrate for measuring the trypsin-like peptidase activity of the 20S proteasome.
Suc-Leu-Leu-Val-Tyr-AMC AG-CP3-0016 Fluorogenic substrate for measuring the chymotrypsin-like peptidase activity of the 20S proteasome, calpains and other chymotrypsin-like proteases.
Suc-Leu-Tyr-AMC AG-CP3-0017 Fluorogenic substrate for measuring the chymotrypsin-like peptidase activity of the 20S proteasome.
Z-Leu-Leu-Leu-AMC AG-CP3-0019 Fluorogenic substrate for measuring the chymotrypsin-like peptidase activity of the 20S proteasome.
Z-Leu-Leu-Glu-AMC AG-CP3-0022 Fluorogenic substrate for measuring the caspase-like activity of the 20S proteasome.
Z-Leu-Arg-Gly-Gly-AMC AG-CP3-0023 Preferred substrate sequence of the human deSUMOylating enzymes SENP6 and SENP7.


Proteasome Complex Modulators 

Product Name PID Product Description
Apcin AG-CR1-3603 APC/C E3 ubiquitin ligase inhibitor.
Auranofin AG-CR1-3611 Proteasomal deubiquitinase (DUB) inhibitor.
BAY 11-7082 AG-CR1-0013 RBR E3 ligase inhibitor. Effects by inactivating the E2-conjugating enzymes Ubc13 and UbcH7 and the E3 ligase LUBAC, preventing the formation of Lys63-linked and linear polyubiquitin chains.
Lovastatin AG-CN2-0051 SKP2 E3 ligase inhibitor.
NSC697923 AG-CR1-3519 Selective Ub-conjugating enzyme (E2) complex Ubc13-Uev1A inhibitor. Inhibits the formation of the Ubc13~Ub conjugate.
Oridonin CDX-O0131 CRL/SCF RING E3 inhibitor. Inhibits Fbw7 an E3 ubiquitin ligase (CRL/SCF RING) of c-Myc and promotes proteasomal degradation.
Simvastatin AG-CN2-0052 SKP2 E3 ligase inhibitor.
Suramin . hexasodium salt AG-CR1-3575 Cullin-RING E3 ubiquitin ligase inhibitor.
Vitexin AG-CN2-0425 Inhibits polyubiquitin synthesis by the ubiquitin-conjugating enzyme E2-25K.


SUMO-Related Inhibitors 

Product Name PID Product Description
Anacardic acid AG-CR1-0046 SUMOylation inhibitor.
MLN4924 [NAE Inhibitor] AG-CR1-3703 Inhibits ubiquitin-activating enzyme (UAE) and SUMO-activating enzyme (SAE) with IC50 values of 1.5 and 8.2μM, respectively.


Proteasome Assay Kits & 20S Proteasome/20S Immunoproteasome Complexes 

The two proteasome kits are designed to test for specific activity of 20S immunoproteasome or 20S constitutive proteasome, and include purified proteasomes, AMC-conjugated substrates, specific inhibitors and necessary buffers and solutions. Fluorescence detection can be performed at Excitation/Emission (nm): 345/445, allowing for a real-time read out of specific activity.

All highly active and pure proteasomes offered by AdipoGen Life Sciences are able to proteolytically degrade substrates in an ATP-independent manner.

Product Name PID Product Description
20S Immunoproteasome Assay Kit SBB-KP0037 Designed to test for specific activity of 20S immunoproteasome.
20S Constitutive Proteasome Assay Kit SBB-KP0038 Designed to test for specific activity of 20S proteasome.
20S Immunoproteasome (human) (untagged) SBB-PP0004 20S immunoproteasome is most active against Suc-LLVY-AMC (AG-CP3-0016), Ac-PAL-AMC (AG-CP3-0036), and Ac-ANW-AMC (AG-CP3-0037) substrates.
20S Immunoproteasome (mouse) (untagged) SBB-PP0083 20S immunoproteasome is most active against Suc-LLVY-AMC (AG-CP3-0016), Ac-PAL-AMC (AG-CP3-0036), and Ac-ANW-AMC (AG-CP3-0037) substrates.
20S Immunoproteasome (rat) (untagged) SBB-PP0046 20S immunoproteasome is most active against Suc-LLVY-AMC (AG-CP3-0016), Ac-PAL-AMC (AG-CP3-0036), and Ac-ANW-AMC (AG-CP3-0037) substrates.
20S Proteasome (human) (untagged) SBB-PP0005 20S Proteasome is most active against Suc-LLVY-AMC (AG-CP3-0016), Z-LLE-AMC (AG-CP3-0022), and Ac-WLA-AMC (AG-CP3-0035) substrates.
20S Proteasome (mouse) (untagged) SBB-PP0047 20S Proteasome is most active against Suc-LLVY-AMC (AG-CP3-0016), Z-LLE-AMC (AG-CP3-0022), and Ac-WLA-AMC (AG-CP3-0035) substrates.
20S Proteasome (rat) (untagged) SBB-PP0086 20S Proteasome is most active against Suc-LLVY-AMC (AG-CP3-0016), Z-LLE-AMC (AG-CP3-0022), and Ac-WLA-AMC (AG-CP3-0035) substrates.
Angiocidin (human) (rec.) AG-40B-0061 Angiocidin shows sequence similarity with proteasome components and is also being referred to as 26A proteasome regulatory subunit S5A.
Ubiquitin (human) (rec.) (Europium-Cryptate) SBB-TR0014 Human ubiquitin (aa1-76) is site-specifically conjugated to a single Europium-Cryptate moiety.
Ubiquitin (human) (rec.) (Cy5) SBB-TR0015 Human ubiquitin (aa1-76) is site-specifically conjugated to a single Cyanine 5 (Cy5) moiety.
Ubiquitin (human) (rec.) (6-FAM) SBB-TR0016 Human ubiquitin (aa1-76) is site-specifically conjugated to a single fluorescein (6-FAM) moiety.


Specialty Degradation Reagents 

Product Name PID Product Description
D-Biotin p-nitrophenyl ester (Biotin-ONP; BNP) CDX-B0307 Exploits the intracellular ubiquitin-proteasome system to selectively degrade target proteins. D-Biotin p-nitrophenyl ester is commonly used as a biotin-tagged photoaffinity probe and an alkyl chain-based PROTAC linker that can be used in the synthesis of PROTACs.
Lipoyl-TRIM21 (human) (rec.) (His) AG-40B-0182 TRIM21 (tripartite motif-containing protein 21) is a cytosolic Fc receptor induced by interferon (IFN). TRIM21 functions as a E3 ligase. During infection, antibodies are delivered efficiently to the cytosol when bound to intracellular pathogens such as viruses and bacteria. The antibody-pathogen complex in the cytosol upon engagement of the protein TRIM21 is ubiquitinylated and degraded by the proteasome machinery.


Buffers and Solutions 

Product Name PID Product Description
AMC Standard Solution SBB-RB0128 A fluorogenic standard useful for quantitating assays monitoring 7-amino-4-methylcoumarin (AMC) release.
AFC Standard Solution SBB-RB0129 A fluorogenic standard useful for quantitating assays monitoring 7-amino-4-trifluoromethylcoumarin (AFC) release.
Loading Buffer (5X) SBB-RB0126 Loading buffer for separation and visualization of proteins with SDS-PAGE and western blot analysis.
MgATP (100X) Solution SBB-RB0127 Pre-coupled Mg-ATP is an ideal energy source for semi-purified conjugation/degradation reactions.
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