Related Experiment Video
Updated: Mar 3, 2026
Detection of Protein Ubiquitination
Published on: August 19, 2009
SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function
Kate E Coleman1, Miklós Békés1, Jessica R Chapman2
1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, United States.
Abstract:
NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to accumulation of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels for CRL-dependent proteostasis.
Insights
This study identifies Ubc12 auto-neddylation and its regulation by SENP8. Loss of SENP8 disrupts neddylation, leading to cell cycle defects and accumulation of cullin-RING ligase substrates, impacting proteostasis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- NEDD8 is a ubiquitin-like modifier crucial for activating cullin-RING ligases (CRLs).
- Identifying true NEDD8 substrates beyond CRLs is challenging due to experimental artifacts from exogenous NEDD8 overexpression.
- Understanding the regulation of neddylation pathway components is vital for cellular proteostasis.
Purpose of the Study:
- To develop a method for stabilizing neddylated proteins to identify novel NEDD8 substrates.
- To investigate the auto-neddylation of Ubc12 and its regulatory mechanisms.
- To elucidate the role of SENP8 in the NEDD8 conjugation pathway and its impact on CRL function.
Main Methods:
- Development of a deconjugation-resistant NEDD8 mutant to stabilize neddylated substrates.
- Identification of Ubc12 as an auto-neddylated substrate using the stabilized NEDD8 approach.
- Characterization of SENP8/DEN1 as the protease deconjugating Ubc12 and analysis of SENP8-deficient cells.
Main Results:
- Ubc12, a NEDD8 E2 enzyme, was identified as a substrate for auto-neddylation.
- SENP8 was identified as the specific protease counteracting Ubc12 auto-neddylation.
- SENP8 deficiency led to aberrant neddylation of pathway components, accumulation of CRL substrates, and impaired cell cycle progression.
Conclusions:
- SENP8 is essential for regulating Ubc12 neddylation and maintaining proper neddylation levels within the pathway.
- Dysregulation of SENP8 impacts CRL-dependent proteostasis and cell cycle control.
- The developed deconjugation-resistant NEDD8 strategy is effective for identifying and validating non-canonical NEDD8 substrates.
Related Concept Videos
00:50Anaphase Promoting Complex
02:58Regulated Protein Degradation
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
02:17Separation of Sister Chromatids
At the onset of anaphase, separase, a proteolytic enzyme, is...
02:04Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein....
01:13The Proteasome
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
02:18The Proteasome
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...