GO:1902523 positive regulation of protein K63-linked ubiquitination: Signaling Pathway, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:1902523 describes any process that activates or increases the frequency, rate or extent of protein K63-linked ubiquitination.
• K63-linked ubiquitination is a non-degradative ubiquitin modification that regulates innate immunity, DNA repair, and oncogenic signaling [2,7].
• Key positive regulators include E3 ligases such as PELI1, TRIM21, and TRIM28, as well as deubiquitinase complexes that indirectly promote K63 chains [1,3,8].
• Dysregulation of K63-linked ubiquitination is linked to breast cancer metastasis, antiviral immunity, and immune checkpoint regulation [1,2,8].
• CRISPR knockout, point mutation, and knock-in models are essential to dissect causal roles of K63-linked ubiquitination enzymes.
• EDITGENE provides end-to-end CRISPR services to study GO:1902523, from library screening to bioinformatics.
Description
GO:1902523, positive regulation of protein K63-linked ubiquitination, is a biological process that increases the attachment of K63-linked polyubiquitin chains to target proteins. Unlike K48-linked chains that typically signal proteasomal degradation, K63-linked ubiquitination serves as a scaffold for protein-protein interactions and signaling complex assembly [2,7]. This process is critical for innate antiviral immunity, where USP18 promotes K63-linked ubiquitination of MAVS to enhance interferon signaling. It also controls immune checkpoint proteins, as TRIM21-mediated K63-linked ubiquitination of PD-1 modulates T cell activity. Researchers study GO:1902523 to understand how cells mount rapid immune responses and how cancer cells hijack these pathways for metastasis [1,8]. The term encompasses the activity of E3 ligases, deubiquitinases, and adaptor proteins that together determine the extent and specificity of K63-linked ubiquitination [1,3,8].
positive regulation of protein K63-linked ubiquitination At A Glance
| GO ID | GO:1902523 |
|---|---|
| GO term | positive regulation of protein K63-linked ubiquitination |
| Ontology | biological_process |
| Synonym | activation of protein K63-linked polyubiquitination; upregulation of protein K63-linked ubiquitination; positive regulation of protein K63-linked polyubiquitination |
| Major function | Increases K63-linked ubiquitin chain attachment to substrate proteins, often to promote signaling complex assembly and non-degradative outcomes [2,7]. |
| Key enzymes | E3 ligases (e.g., PELI1, TRIM21, TRIM28) and deubiquitinases (e.g., USP18) [1,2,3,8]. |
| Cellular contexts | Innate immunity, DNA damage response, cancer metastasis, and immune checkpoint regulation [1,2,7,8]. |
| Research methods | CRISPR knockout screens, ubiquitination assays, mass spectrometry, and imaging. |
What Is GO:1902523?
Positive regulation of protein K63-linked ubiquitination (GO:1902523) refers to any cellular process that activates or increases the frequency, rate, or extent of attaching K63-linked ubiquitin chains to a protein substrate. This regulation can occur through increased activity of E3 ubiquitin ligases, enhanced recruitment of ubiquitin-conjugating enzymes, or inhibition of deubiquitinases that remove K63 chains [2,8]. The process is distinct from K48-linked ubiquitination because K63 chains do not target proteins for degradation but instead serve as signaling platforms [2,7].
Why Is positive regulation of protein K63-linked ubiquitination Important in Cell Biology?
Understanding positive regulation of K63-linked ubiquitination is crucial because this process controls key signaling hubs in immunity and cancer [2,8]. For example, USP18 promotes K63-linked ubiquitination of MAVS to sustain antiviral responses, and loss of this regulation impairs host defense. In cancer, PELI1 and EGFR cooperate to enhance K63-linked ubiquitination events that drive breast cancer metastasis. TRIM21-mediated K63-linked ubiquitination of PD-1 regulates immune checkpoint blockade efficacy, linking this process to immunotherapy outcomes. Thus, GO:1902523 is a focal point for therapeutic strategies targeting ubiquitin signaling [1,8].
• Controls innate antiviral immunity by stabilizing MAVS signaling complexes.
• Promotes breast cancer metastasis through PELI1-EGFR cooperation.
• Regulates PD-1 stability and immune checkpoint blockade response.
• Modulates STING trafficking and termination of immune signaling.
• Influences ferroptosis via TRIM28 and ACSL4 regulation.
• Affects sensitivity to immunotherapy through PD-L1 ubiquitination.
• Plays a role in cisplatin resistance via USP1 and MAST1.
• Involved in esophageal squamous cell carcinoma through USP10 and ANLN.
• Provides targets for CRISPR-based functional genomics.
• Offers opportunities for therapeutic intervention in cancer and infectious diseases [1,8].
What Happens During positive regulation of protein K63-linked ubiquitination?
Recognition and recruitment of E3 ligases
In simple terms: First, the cell brings the right enzyme to the target protein.
Positive regulation begins when specific E3 ubiquitin ligases are recruited to substrate proteins. For instance, PELI1 acts as an E3 ligase that promotes K63-linked ubiquitination of target proteins in breast cancer cells. Similarly, TRIM21 functions as an E3 ligase that adds K63-linked ubiquitin chains to PD-1, thereby regulating its function. This step often requires adaptor proteins or post-translational modifications that create docking sites for the ligase [1,8].
Assembly of K63-linked ubiquitin chains
In simple terms: Then, a chain of ubiquitin molecules is built on the target.
Once the E3 ligase is engaged, it collaborates with E2 conjugating enzymes to attach ubiquitin molecules via lysine 63 linkages. USP18 promotes K63-linked polyubiquitination of MAVS, enhancing its signaling capacity. This chain assembly is not degradative; instead, it creates a platform for downstream signaling complexes [2,7].
Deubiquitinase modulation
In simple terms: Brakes are removed to allow more chain building.
Positive regulation can also occur by inhibiting deubiquitinases that would otherwise remove K63 chains. For example, targeting USP10 induces degradation of ANLN, but in other contexts, deubiquitinases like USP1 regulate MAST1-driven cisplatin resistance [5,6]. The balance between ligases and deubiquitinases determines the net level of K63-linked ubiquitination [5,6].
Downstream signaling activation
In simple terms: Finally, the chain sends a signal inside the cell.
K63-linked ubiquitin chains serve as scaffolds that recruit signaling molecules. In STING signaling, K63-linked ubiquitination events are terminated through ESCRT-dependent microautophagy, showing that positive regulation must be tightly controlled. In immune cells, TRIM21-mediated K63-linked ubiquitination of PD-1 modulates T cell receptor signaling and checkpoint blockade efficacy.
Key Genes Involved in GO:1902523 positive regulation of protein K63-linked ubiquitination
The following genes and proteins are experimentally validated participants in positive regulation of protein K63-linked ubiquitination (GO:1902523).
| Gene | Major Role | Research Relevance |
|---|---|---|
| PELI1 | E3 ligase promoting K63-linked ubiquitination | Breast cancer metastasis |
| USP18 | Promotes K63-linked polyubiquitination of MAVS | Innate antiviral immunity |
| TRIM28 | Promotes SUMOylation and inhibits OPTN-selective autophagic degradation of ACSL4 | Neuronal ferroptosis |
| TRIM21 | E3 ligase mediating K63-linked ubiquitination of PD-1 | Immune checkpoint blockade and CAR-T therapy |
| MAVS | Substrate of K63-linked ubiquitination | Antiviral signaling |
| PD-1 | Substrate of K63-linked ubiquitination | T cell exhaustion and immunotherapy |
| PD-L1 | Regulated by Skp2-mediated ubiquitination | Immunotherapy sensitivity in NSCLC |
| ANLN | Target of USP10-mediated degradation | Esophageal squamous cell carcinoma |
| MAST1 | Regulated by USP1 | Cisplatin resistance |
| STING | K63-linked ubiquitination in trafficking | Immune signaling termination |
| EGFR | Cooperates with PELI1 | Breast cancer metastasis |
| ACSL4 | Regulated by TRIM28 | Ferroptosis |
| OPTN | Autophagic receptor | Neuronal ferroptosis |
| Skp2 | E3 ligase for PD-L1 | NSCLC immunotherapy |
| USP10 | Deubiquitinase | ESCC |
| USP1 | Deubiquitinase | Cisplatin resistance |
| ESCRT | Complex mediating microautophagy | STING degradation |
How Is positive regulation of protein K63-linked ubiquitination Regulated?
Positive regulation of K63-linked ubiquitination is controlled at multiple levels. E3 ligases such as PELI1 and TRIM21 are themselves regulated by phosphorylation and protein-protein interactions [1,8]. Deubiquitinases like USP18 and USP10 can either promote or reverse K63-linked ubiquitination depending on context [2,5]. Additionally, redox regulation of TRIM28 influences its ability to promote SUMOylation and inhibit autophagic degradation of ACSL4, indirectly affecting K63-linked ubiquitination dynamics. The STING pathway is terminated by ESCRT-dependent microautophagy, illustrating spatial and temporal control of K63-linked ubiquitination.
positive regulation of protein K63-linked ubiquitination and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PELI1 | Breast cancer metastasis | Knockout in MDA-MB-231 cells |
| USP18 | Antiviral immunity | Knockout in macrophages |
| TRIM28 | Neuronal ferroptosis | Point mutation in neurons |
| TRIM21 | Immune checkpoint blockade | Knock-in of tagged PD-1 |
| Skp2 | NSCLC immunotherapy | Overexpression in lung cancer cells |
Cancer metastasis
PELI1 and EGFR cooperate to promote K63-linked ubiquitination events that drive breast cancer metastasis. Targeting this axis may reduce metastatic spread. In non-small cell lung cancer, Skp2-mediated ubiquitination of PD-L1 dictates sensitivity to immunotherapy. These findings link GO:1902523 to tumor progression and treatment response.
Antiviral immunity
USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS. This modification enhances MAVS signaling and interferon production. Dysregulation of this process can lead to impaired antiviral defense, highlighting the importance of GO:1902523 in infectious disease.
Neurodegeneration and ferroptosis
Redox regulation of TRIM28 facilitates neuronal ferroptosis by promoting SUMOylation and inhibiting OPTN-selective autophagic degradation of ACSL4. This pathway intersects with K63-linked ubiquitination networks, suggesting roles in neurodegenerative conditions.
Immune checkpoint regulation
Targeting the TRIM21-PD-1 axis potentiates immune checkpoint blockade and CAR-T cell therapy. TRIM21-mediated K63-linked ubiquitination of PD-1 modulates T cell activity, making GO:1902523 a therapeutic target in immuno-oncology.
From positive regulation of protein K63-linked ubiquitination-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does PELI1 promote K63-linked ubiquitination in breast cancer? | PELI1 knockout via CRISPR |
| Does USP18 regulate MAVS ubiquitination? | USP18 knockout in immune cells |
| Does TRIM21 mediate PD-1 ubiquitination? | TRIM21 knockout or point mutation |
| Does TRIM28 redox regulation affect ferroptosis? | TRIM28 point mutation knock-in |
| Does Skp2 regulate PD-L1 ubiquitination? | Skp2 overexpression |
| Does USP1 regulate MAST1-driven cisplatin resistance? | USP1 knockout |
How to Study the positive regulation of protein K63-linked ubiquitination Process
| Method | What It Measures | Typical Application |
|---|---|---|
| CRISPR knockout screen | Gene essentiality for K63-linked ubiquitination | Identify novel regulators |
| In vivo ubiquitination assay | K63-linked chain formation on substrate | Validate E3 ligase activity |
| Mass spectrometry | Ubiquitination sites and chain topology | Map modifications on MAVS, PD-1 [2,8] |
| Immunofluorescence | Subcellular localization of ubiquitinated proteins | Track STING trafficking |
| Co-immunoprecipitation | Protein-protein interactions | Detect E3-substrate complexes |
| Western blot with linkage-specific antibody | K63-linked ubiquitin levels | Quantify changes in knockout cells |
| RNA-seq | Transcriptional changes | Assess downstream effects |
| Flow cytometry | Surface PD-1 or PD-L1 levels | Measure immune checkpoint regulation |
CRISPR knockout screens
Genome-wide CRISPR knockout screens can identify deubiquitinases and E3 ligases that regulate K63-linked ubiquitination. For example, a CRISPR/Cas9-based screen identified USP1 as a regulator of MAST1-driven cisplatin resistance. This approach is powerful for discovering novel positive regulators within GO:1902523.
Ubiquitination assays
In vivo and in vitro ubiquitination assays using tagged ubiquitin mutants (K63-only) can measure the extent of K63-linked chain formation on substrates like MAVS or PD-1 [2,8]. These assays often use immunoprecipitation followed by immunoblotting with linkage-specific antibodies.
Mass spectrometry
Mass spectrometry-based proteomics can map ubiquitination sites and quantify K63-linked chains on target proteins. This method has been used to identify modifications on MAVS and PD-1 [2,8].
Imaging and trafficking studies
Fluorescence microscopy can track the localization of K63-linked ubiquitinated proteins and their trafficking through endosomes, as shown for STING. Live-cell imaging reveals dynamic regulation of GO:1902523.
How CRISPR Can Be Used to Study GO:1902523 positive regulation of protein K63-linked ubiquitination
Knockout
CRISPR knockout of E3 ligases or deubiquitinases can abolish positive regulation of K63-linked ubiquitination. For example, USP1 knockout sensitizes cancer cells to cisplatin by affecting MAST1. Knockout models are essential to establish causality in GO:1902523.
Point Mutation
Point mutations can disrupt catalytic activity or specific interaction domains. For instance, mutating the RING domain of TRIM21 would prevent its E3 ligase activity, blocking PD-1 ubiquitination. Such models help dissect domain-specific functions.
Knock-in
Knock-in of tagged ubiquitin or substrate proteins allows tracking of K63-linked chains in live cells. Tagged PD-1 knock-in can reveal real-time ubiquitination dynamics. This approach is valuable for imaging studies.
Overexpression
Overexpression of E3 ligases like PELI1 or TRIM21 can enhance K63-linked ubiquitination and drive phenotypes such as metastasis [1,8]. Overexpression models are useful for gain-of-function studies.
How EDITGENE Supports positive regulation of protein K63-linked ubiquitination Research
Researchers studying positive regulation of protein K63-linked ubiquitination-related genes often need to determine whether a candidate gene is causally involved in chain assembly, substrate selection, or downstream signaling. EDITGENE provides validated CRISPR tools and services to accelerate these investigations.
Contact EDITGENE today to design your custom CRISPR model for positive regulation of protein K63-linked ubiquitination research.
Frequently Asked Questions About positive regulation of protein K63-linked ubiquitination
What is GO:1902523?
GO:1902523 is the Gene Ontology term for positive regulation of protein K63-linked ubiquitination, describing processes that increase K63-linked ubiquitin chain attachment to proteins.
What genes are involved in positive regulation of protein K63-linked ubiquitination?
Key genes include PELI1, USP18, TRIM21, TRIM28, and their substrates such as MAVS and PD-1 [1,2,3,8].
How does K63-linked ubiquitination differ from K48-linked ubiquitination?
K63-linked chains are non-degradative and serve as signaling scaffolds, while K48-linked chains typically target proteins for proteasomal degradation [2,7].
What diseases are associated with K63-linked ubiquitination?
It is linked to breast cancer metastasis, antiviral immunity, ferroptosis, and immune checkpoint regulation [1,2,3,8].
How can I study positive regulation of K63-linked ubiquitination?
Use CRISPR knockout screens, ubiquitination assays, mass spectrometry, and imaging.
What is the role of USP18 in K63-linked ubiquitination?
USP18 promotes K63-linked polyubiquitination of MAVS to enhance innate antiviral immunity.
How does TRIM21 regulate PD-1?
TRIM21 mediates K63-linked ubiquitination of PD-1, affecting immune checkpoint blockade and CAR-T therapy.
What experimental models are used for GO:1902523?
Knockout, point mutation, knock-in, and overexpression models in cancer and immune cells [1,6,8].
Can CRISPR screens identify regulators of K63-linked ubiquitination?
Yes, a CRISPR/Cas9 screen identified USP1 as a regulator of MAST1-driven cisplatin resistance.
What services does EDITGENE offer for ubiquitination research?
EDITGENE provides knockout, point mutation, knock-in, overexpression, library screening, and bioinformatics services.
Conclusion
GO:1902523, positive regulation of protein K63-linked ubiquitination, is a central process in immune signaling and cancer biology. Its dysregulation contributes to metastasis, antiviral responses, and immunotherapy outcomes [1,2,8]. CRISPR-based models are indispensable for dissecting the causal roles of E3 ligases and deubiquitinases in this pathway. EDITGENE offers comprehensive services to support your research on K63-linked ubiquitination.
References
- 1. Qi J et al.. 2023. PELI1 and EGFR cooperate to promote breast cancer metastasis.. Oncogenesis 12(1):9 PMID: 36841821
- 2. Hou J et al.. 2021. USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS.. Nat Commun 12(1):2970 PMID: 34016972
- 3. Liu W et al.. 2025. Redox regulation of TRIM28 facilitates neuronal ferroptosis by promoting SUMOylation and inhibiting OPTN-selective autophagic degradation of ACSL4.. Cell Death Differ 32(6):1041-1057 PMID: 39875520
- 4. Lv L et al.. 2024. LKB1 dictates sensitivity to immunotherapy through Skp2-mediated ubiquitination of PD-L1 protein in non-small cell lung cancer.. J Immunother Cancer 12(12) PMID: 39694700
- 5. Cao YF et al.. 2023. Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma.. Cell Death Differ 30(2):527-543 PMID: 36526897
- 6. Tyagi A et al.. 2022. CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.. Theranostics 12(13):5949-5970 PMID: 35966591
- 7. Kuchitsu Y et al.. 2023. STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes.. Nat Cell Biol 25(3):453-466 PMID: 36918692
- 8. Shi J et al.. 2025. Targeting the TRIM21-PD-1 axis potentiates immune checkpoint blockade and CAR-T cell therapy.. Mol Ther 33(3):1073-1090 PMID: 39905727
