Ubiquitin-Proteasome System (UPS) Assays and Profiling

CD Biosynsis offers comprehensive Ubiquitin-Proteasome System (UPS) Assays critical for drug discovery programs targeting protein turnover, a central regulator of cellular life and disease (oncology, neurodegeneration). The UPS controls protein degradation through a precise cascade involving E1, E2, E3 enzymes, and the 26S Proteasome. Our platform provides quantitative measurement of enzymatic activity and inhibitor potency (IC50, Ki) across all major UPS components. We employ highly sensitive, high-throughput technologies, including fluorescence-based cascade assays, DUB activity assays, and Proteasome subunit activity measurements. Researchers can select and combine individual component modules to build a custom profiling panel, enabling precise characterization of compound effects on ubiquitination, deubiquitination, and proteasomal degradation.

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Enzymatic and Functional Characterization of Protein Turnover

Targeting the Ubiquitin-Proteasome System (UPS) requires precise mechanistic insight, as therapeutic success depends on selectively modulating one component (e.g., a specific E3 Ligase or DUB) without causing global toxicity. Our service is built on validated, recombinant enzymes and robust cellular assays. We provide selectable panels allowing you to focus on the cascade step most relevant to your drug candidate: from the initiation step (E1 activation), through polyubiquitin chain synthesis (E2/E3), to the final removal of the ubiquitin tag (DUBs), or the degradation motor (Proteasome). This focused, customizable approach delivers the high-quality kinetic data necessary to understand inhibitor mechanism and validate therapeutic specificity.

Customizable Ubiquitin-Proteasome Pathway Assays

E1, E2, and E3 Ligase Assays DUB (Deubiquitinase) Assays Proteasome and Functional Assays

E1, E2, and E3 Ligase Component Profiling

Choose Specific Components for Ubiquitination Assays

Check the box next to the UPS component you wish to profile for activity or inhibition:

E1 Activating Enzyme Activity (UBA1)

E2 Conjugating Enzyme Activity (Selectable Panel)

E3 Ligase Substrate Ubiquitination (Selectable Targets)

ISGylation Cascade Assays

In Vitro Ubiquitin Chain Synthesis (K48, K63)

SUMOylation Cascade Assays

Neddylation Cascade Assays (NEDD8)

All Three Cascade Assays (Ub/SUMO/NEDD8)

DUB (Deubiquitinase) Enzyme Profiling

Choose Key DUB Targets for Activity and Selectivity

Select the specific DUB targets you require for activity and inhibitor screening (IC50, Ki):

USP7 (Target for Oncology)

UCH-L1 (Target for Neurodegeneration)

BAP1 (Tumor Suppressor)

OTUB1/OTUB2

CYLD

USP2

USP14/UCH37 (Proteasome-associated)

Full DUB Selectivity Panel (40+ DUBs)

Proteasome and Functional Assays

Select Terminal Degradation and Functional Readouts

20S/26S Proteasome Activity

Measurement of chymotrypsin-like, trypsin-like, and caspase-like activities using fluorogenic substrates.

Proteasome Subunit Specificity

IC50 determination against individual catalytic subunits of the 20S core particle (e.g., PSMB5).

Cellular Ubiquitin Pool Analysis

Western Blot or ELISA quantification of global polyubiquitinated protein levels in inhibitor-treated cells.

Ubiquitin-Proteasome System Profiling Workflow

A multi-step approach for cascade characterization.

Component Selection and Enzyme Sourcing

Assay Optimization and IC50 Profiling

Kinetic Analysis and Selectivity Testing

Functional and Mechanistic Reporting

Target Panel Customization: Finalize the list of E1, E2, E3, DUB, and Proteasome targets.

Enzyme QC: Verify the activity and purity of all recombinant components (including substrate-Ubiquitin conjugates).

Assay Setup: Implement the appropriate detection method (e.g., TR-FRET, coupled assay, or fluorescence release) for each component.

Potency Screening: Perform dose-response curves for inhibitors against the custom-selected panel to calculate IC50 values.

Ki Determination: Conduct comprehensive kinetic analysis (varying substrate/ATP/inhibitor) to determine the inhibition constant (Ki) and MoA.

Selectivity Index: Profile lead compounds against closely related targets (e.g., DUBs vs. other DUBs) to ensure specificity.

  • Proteasome Analysis: Measure inhibition of all three catalytic activities (Chymotrypsin, Trypsin, Caspase) in the 20S or 26S complex.
  • Cellular Confirmation: Use Western Blot to confirm the accumulation of polyubiquitinated proteins in cell culture (if selected).
  • Reporting: Deliver comprehensive reports including raw data, analyzed plots, IC50/Ki values, and mechanistic interpretation.

Specialized Assays for the UPS Cascade

Modular Cascade Profiling

           

Ability to test inhibition across E1, E2, E3, DUBs, and the Proteasome for full pathway MoA mapping.

Extensive DUB/E3 Ligase Library

           

Access to one of the industry's largest collections of recombinant DUB and E3 enzymes for high-specificity screening.

Precise Mechanistic Kinetics

           

Accurate determination of Ki, inhibition mode, and time-dependent inhibition for challenging enzymes.

Proteasome Subunit Specificity

           

Ability to separately measure the three catalytic activities (Chymotrypsin, Trypsin, Caspase) of the 20S core.

Client Testimonials on UPS Assays

"The DUB selectivity profiling across 40+ enzymes was critical. It confirmed that our lead compound was highly specific for USP7, guiding our lead optimization efforts."

Dr. Jian Li, Cancer Biology Research

"The in vitro E3 Ligase assay, coupled with K63 chain synthesis, provided definitive evidence of our compound's mechanism of enhancing specific ubiquitination."

Ms. Anya Sharma, Therapeutic Development Lead

"The detailed kinetic profiling on the 20S Proteasome, measuring all three activities, confirmed our inhibitor was highly potent and specific for the chymotrypsin-like site."

Mr. Julian Chen, Preclinical Program Manager

FAQs about Ubiquitin-Proteasome System (UPS) Assays

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What are the three main enzymatic steps in ubiquitination?

The three main enzymatic steps are: activation by the E1 ubiquitin-activating enzyme, conjugation by the E2 ubiquitin-conjugating enzyme, and ligation by the E3 ubiquitin ligase.

What is the function of Deubiquitinases (DUBs)?

DUBs remove ubiquitin tags from target proteins, regulating the stability of the substrate and recycling free ubiquitin. They often oppose the action of E3 ligases.

How do you distinguish between K48 and K63 ubiquitin chains?

We use recombinant E2/E3 enzymes that are specific for either K48-linked (degradation signal) or K63-linked (signaling/non-proteolytic) polyubiquitin chain formation in in vitro cascade assays.

What are the three activities measured in the 20S Proteasome?

The three main activities measured are: chymotrypsin-like, trypsin-like, and caspase-like activities, corresponding to the cleavage preference of the three distinct catalytic beta subunits (PSMB5, PSMB2, and PSMB7).

How much does Metabolic Engineering services cost?

The cost of Metabolic Engineering services depends on the project scope, complexity of the target compound, the host organism chosen, and the required yield optimization. We provide customized quotes after a detailed discussion of your specific research objectives.

Do your engineered strains meet regulatory standards?

We adhere to high quality control standards in all strain construction and optimization processes. While we do not handle final regulatory approval, our detailed documentation and compliance with best laboratory practices ensure your engineered strains are prepared for necessary regulatory filings (e.g., GRAS, FDA).

What to look for when selecting the best gene editing service?

We provide various gene editing services such as CRISPR-sgRNA library generation, stable transformation cell line generation, gene knockout cell line generation, and gene point mutation cell line generation. Users are free to select the type of service that suits their research.

Does gene editing allow customisability?

Yes, we offer very customised gene editing solutions such as AAV vector capsid directed evolution, mRNA vector gene delivery, library creation, promoter evolution and screening, etc.

What is the process for keeping data private and confidential?

We adhere to the data privacy policy completely, and all customer data and experimental data are kept confidential.

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