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Article

The Degradation Pathway of COP9 Signalosome–Cullin-RING Ubiquitin Ligase Complexes via Autophagy

by
Dawadschargal Dubiel
1,*,
Roland Hartig
2 and
Wolfgang Dubiel
1,*
1
Institute of Experimental Internal Medicine, Medical Faculty, Otto Von Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany
2
Multi-Parametric Bioimaging and Cytometry Unit (Confocal Microscopy & Flow Cytometry), Institute of Molecular and Clinical Immunology, Medical Faculty, Otto Von Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany
*
Authors to whom correspondence should be addressed.
Biomolecules 2026, 16(2), 218; https://doi.org/10.3390/biom16020218
Submission received: 2 December 2025 / Revised: 12 January 2026 / Accepted: 19 January 2026 / Published: 2 February 2026
(This article belongs to the Section Molecular Biology)
Browse Figures
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Abstract

In Mammalia, the COP9 signalosome (CSN) is associated with cullin-RING ubiquitin ligases (CRLs). This study focuses on the variants CSNCSN7A and CSNCSN7B, which form complexes with CRL3 and CRL4A, respectively. Although some research has been conducted on the assembly of the complexes, little is known about their breakdown. Here, we show that entire CSNCSN7A-CRL3 and CSNCSN7B-CRL4A complexes are degraded via autophagy. CSN-CRL complexes are degraded in the absence of serum via bulk autophagy and in the presence of the specific inhibitor of CSN, CSN5i-3, via selective macroautophagy. Surprisingly, the self-ubiquitylation of cullins in the CRLs was identified as a specific signal for selective macroautophagy. The self-ubiquitylation of cullins takes place in the presence of CSN5i-3, and CSN-CRL complexes are expelled from the nucleus to be degraded in the cytosol. Selective macroautophagy can be blocked by chloroquine, a specific inhibitor of autophagy. Interestingly, the process can also be inhibited by MLN4924, a neddylation inhibitor. Confocal fluorescence microscopy illustrates the interaction of CSN subunits with ATG8, as well as with RAB7, both in HeLa and in LiSa-2 cells. Confocal fluorescence microscopy produces images that suggest the localization of CSN-CRL particles in autophagosomes. Our data place CSN-CRL in the category of large complexes that are degraded through autophagy.

1. Introduction

The COP9 signalosome (CSN) belongs to a group of paralog particles called ZOMES, complexes that include the 26S proteasome LID and the eukaryotic initiation factor 3 (eIF3). In Mammalia, the core CSN consists of six Proteasome lid-CSN-Initiation factor 3 (PCI) domain subunits (CSN1-CSN4, CSN7, and CSN8) and of two MPR1/PAD1 N-terminal (MPN) domain (CSN5 and CSN6) subunits [1]. The CSN is more heterogeneous than indicated by its eight-core-subunit structure [2]. A fraction of the cellular CSN contains a non-canonical subunit [3,4]. Many human CSNs are associated with deubiquitylating enzymes (DUBs) [5] or proteins like p27 and p53 [1]. CSN subunits exist as paralogs forming CSN variants. We are focused on CSN7, which is expressed by the paralog COPS7A/CSN7A, making the CSN variant CSNCSN7A and the paralog COPS7B/CSN7B, which occurs in the CSN variant CSNCSN7B. The variants CSNCSN7A and CSNCSN7B appear simultaneously in most human cells. The CSN is a multi-DUB complex [5] removing NEDD8 from the cullins of cullin-RING ubiquitin ligases (CRLs) [2,5,6,7]. The CSN is associated with the CRL complex, and interaction between the two is the reason for conformational changes to CSN2, CSN4, and CSN7, which move the CSN5-CSN6 dimer into position for deneddylation [6,8,9]. The aforementioned CSN variants and specific CRLs form permanent complexes that represent a reservoir of different cellular functions [1,10]. CSNCSN7A interacts with CRL3, and CSNCSN7B forms permanent complexes with CRL4A. The complexes CSNCSN7A-CRL3 and CSNCSN7B-CRL4A exist side by side in all studied cells [1]. To be active, CSN-CRL complexes need substrate receptors (SRs) and the neddylation of cullins. The appropriate SRs occur in a manner that is dependent on the available substrates, and neddylation is a highly regulated process [1,11]. Under conditions of differentiation, such as during adipogenesis, SRs are mostly exchanged quickly using cullin-associated and neddylation-dissociated protein 1 (CAND1) [1].
Little is known about the turnover of CSN-CRL complexes; however, the assembly of fungal CSN from two trimeric intermediates was recently published [12]. These novel findings provide insight into the assembly of CRL1 and CRL3 complexes [13,14,15], and the accompanying electron microscopy images present views of CSN-CRL interaction [6]. However, nothing is known about CSN-CRL catabolism, though recent data indicate that degradation may take place via autophagy [16,17]. Some large protein complexes, like the 26S proteasome [18,19,20] and CDC48 [21], are degraded via autophagy, in a process in which both nutrient starvation and 26S proteasome inhibition lead to autophagy, also called proteaphagy, implying that bulk and selective routes for using the ubiquitylation of RPN10 as an autophagic receptor, tethering the complex to ATG8, do exist [18,19].
In this study, we show that CSN-CRL is degraded via autophagy as an entire complex. Whether via bulk autophagy, using serum starvation, or selective macroautophagy, induced by a CSN5i-3 inhibitor, CSN-CRL complexes are finally degraded by lysosomes. In the event of selective macroautophagy, CSN-CRL complexes are first tethered to ATG8 by phagophores prior to autophagy. The self-ubiquitylation of cullins in CRL complexes serves as a trigger, and can be inhibited through blocking neddylation.

2. Materials and Methods

2.1. Generation of Stable Cell Lines

The generation of FLAG, FLAG-CSN7A, and FLAG-CSN7B plasmids has been described previously [1]. The HA-tagged ubiquitin (HA-Ub) plasmid was purchased from Addgene (Watertown, MA, USA).
To generate stable transfectants, the HA-Ub plasmid was transfected into HeLa cells using Lipofectamine 2000 (ThermoFisher, Waltham, MA, USA), according to the manufacturer’s protocol. After transfection, HeLa cells permanently expressing HA-Ub were obtained via 0.5 mg/mL G418 administration for 1 to 3 weeks. HA-Ub-containing individual clones were isolated and propagated in the selection medium. CSN7A and CSN7B knockout cells were used and were generated using CRISPR-Cas9 technology as described previously [1].

2.2. RNA Interference

SiRNA transfection against GFP (control) and ATG7 was performed with the Lipofectamine 2000 transfection reagent (ThermoFisher) according to the manufacturer’s protocol. The transfection of siRNAs into HeLa cells was performed at a final concentration of 100 nM of siRNA, 72 h prior to further experiments.

2.3. Protein Extraction, Immunoblotting, and Immunoprecipitation

Cell lysates were obtained in triple-detergent buffer (50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 0.02% (w/v) sodium azide, 0.1% (w/v) SDS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate) as outlined previously [1]. Western blots with appropriate samples were performed and analyzed as described [1].
The mono-detergent lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100) was outlined previously [1]. For denaturing, cells were lysed with triple-detergent lysis buffer with 1% SDS and heated at 95 °C for 10 min. Then, the samples were diluted to a ratio of 1:10 with triple-detergent buffer. Cellular debris was removed via centrifugation for 10 min at 13.000 rpm and 4 °C. Appropriate antibodies, incubated with supernatants, were used at 1–5 µg for 2 h at 4 °C. Protein-A Sepharose was used, and the mixture was rotated overnight at 4 °C. After washing the beads three times with the appropriate lysis buffer, they were boiled with 1× Laemmli SDS-PAGE sample buffer. Immunocomplexes were separated on SDS-PAGE and analyzed via immunoblotting with the following antibodies: anti-CSN3 (Abcam, Cambridge, UK), anti-CSN5 (Cell Signaling, Danvers, MA, USA), anti-CSN7A (Santa Cruz, Santa Cruz, CA, USA), anti-CSN7B (Abcam, Cambridge, UK), anti-CUL3 (BD biosciences, San Jose, CA, USA), anti-CUL4A (Abcam), anti-CDC48 (Abcam), anti-FLAG (Sigma, St. Louis, MO, USA), anti-HA (Santa Cruz), anti-NEDD8 (Invitrogen, Carlsbad, CA, USA), anti-C23 (Santa Cruz), anti-RAB7 (Santa Cruz), anti-Ubiquitin (Santa Cruz), p62 (Abcam), TOLLIP (Abcam), and anti-γ-tubulin (Santa Cruz).

2.4. Density Gradient Centrifugation

As described above, HeLa cells (107) untreated and treated with 1 μM of CSN5i-3 were lyzed with mono-detergent lysis buffer. Cell extracts were loaded onto a 10–30% density glycerol gradient, and ultracentrifugation (27,000 rpm for 24 h at 4 °C) was performed as described [22]. Following centrifugation, the gradients were fractionated into 600 μL volumes. Each fraction (10 μL) was analyzed via SDS–PAGE and immunoblotting with different antibodies. CDC48 served as a marker protein.

2.5. FLAG Pulldowns

LiSa-2 cells with FLAG-tagged CSN7A paralogs were lyzed in mono-detergent buffer as outlined above. Cell lysates were loaded into a pre-equilibrated ANTI-FLAG M2 affinity column (Sigma, St. Louis, MO, USA). Following washing with 20 column volumes of mono-detergent lysis buffer, bound proteins were eluted through competition with 100 µg/mL of the FLAG peptide, as recommended by the manufacturer’s protocol. SDS-PAGE was used to separate proteins, which were later analyzed via immunoblotting with the indicated antibodies.

2.6. Subcellular Fractionation

The fractionation of subcellular compartments has previously been outlined [1].

2.7. Confocal Fluorescence Microscopy and Immunostaining

HeLa and LiSa-2 cells with different backgrounds were exposed in chamber slides. After treatment with different inhibitors, the cells were washed three times with PBS and fixed in 4% paraformaldehyde for 15 min. Fixed samples were permeabilized with 0.1% TritonX-100/PBS for 10 min at room temperature and blocked with 3% filtered BSA in PBS for 1 h. The samples were incubated with primary antibodies directed against LAMP2 (rabbit), ATG8 (rabbit), RAB7 (mouse), CSN3 (rabbit), or CSN7A (mouse) in the indicated combinations in 1% BSA in PBS overnight at 4 °C. Thereafter, the samples were washed three times in PBS and stained for 2 h at room temperature with secondary antibodies coupled to FITC 647 (anti-rabbit) and Cy3 (anti-mouse) fluorophores with 1% BSA in PBS. The cell nuclei were counterstained using DAPI. The slides were visualized and analyzed as described previously [1].

2.8. Statistics

Bands from SDS PAGE were visualized with a ChemoCam Imager (Intas, Göttingen, Germany) and quantified using ImageJ software (version 1.51d). GraphPad Prism 8.0.1 software was used to calculate statistical significance. Error bars indicate standard deviations (SDs). For statistical analysis, unpaired Student’s t-tests were applied. n represents the number of independent experiments. Statistical details of individual experiments can be found in figure legends.

3. Results

3.1. The COP9 Signalosome Is Degraded as a Complex upon Serum Starvation and Specific Inhibition via the CSN5i-3 Inhibitor

In LiSa-2 cells, serum starvation and inhibition by the deneddylation inhibitor CSN5i-3 [23] led to the degradation of the whole CSN protein complex consisting of eight subunits, most likely via autophagy. Control subunits of the 26S proteasome, RPN1 and 20Sa4, were not influenced by CSN5i-3 (1 µM) (Figure 1A). The selected CSN subunits CSN3, CSN5, CSN7A, and CSN7B were characterized by a maximum degradation of 20% following 48 h of starvation. In contrast, the presence of CSN5i-3 led to degradation greater than 50% after 48 h of treatment (Figure 1B). In CSN7A or CSN7B, knockout HeLa cells brought about a similar degradation of CSN subunits as in wild-type (WT) cells (Figure 1C). We conclude that both CSNCSN7A and CSNCSN7B are degraded in a manner that is similar to whole-protein complexes in the presence of CSN5i-3. Figure 1D shows that CSN variants equipped with FLAG-CSN7A or FLAG-CSN7B in LiSa-2 cells were also degraded in a manner dependent on the CSN5i-3 inhibitor concentration, probably via autophagy. A concentration of 0.5 µM CSN5i-3 was sufficient to trigger degradation. Moreover, as shown in Figure 1E, degradation of CSN was induced by 1 µM CSN5i-3 after 28 h in all studied cells, making the process universal. We selected these conditions for further experiments.

3.2. The COP9 Signalosome Is Degraded in Association with Cullin-Ring Ubiquitin Ligases

In human cells, permanent CSN-CRL complexes are localized predominantly to the nucleus [1] due to their functions as cell cycle regulators or chromatin protectors [24]. Using 1 µM CSN5i-3, both CSN subunits as well as CUL3 and CUL4A were expelled from the nucleus (Figure 2A). This was the case in both HeLa and in LiSa-2 cells and indicated that the CSN subunits and cullins had been subjected to similar processes. To ensure that both CSN subunits and cullins were degraded together, we calculated ratios between the CUL4A and CSN subunits upon CSN3 immunoprecipitations before and after the stimulation of degradation (Figure 2B, IP: CSN3). Figure 2C demonstrates that the CUL4A/CSN ratio in the CSN3 immunoprecipitations is similar before and after 28 h of degradation in the presence of 1 µM CSN5i-3, indicating equal degradation rates for CUL4, CSN3, and CSN7B. CUL3 immunoprecipitates confirmed the degradation rates of input and CSN3 immunoprecipitates (Figure 2B). The glycerol gradient in Figure 2D demonstrates that CSN-CRL complexes did not decay during 28 h of stimulation in the presence of 1 µM CSN5i-3. They were not dissected into individual parts and seem to have been degraded as an entire complex. As a control for the density gradient, we used CDC48, which migrated in fractions of 500–600 kDa. The fluorescence microscopy images in Figure 2E illustrate once more how CSN-CRL complexes were expelled from the nucleus in the presence of 1 µM CSN5i-3, where they bound with LAMP2, one of the lysosome-associated membrane glycoproteins [25]. The data indicates that CSN-CRL complexes were exported from the nucleus to the cytosol together, where the autophagy of the entire complex took place.

3.3. COP9 Signalosome–Cullin-Ring Ubiquitin Ligases (CSN-CRLs) Are Degraded as a Whole Complex via Autophagy

As expected from previous experiments, the degradation of CSN-CRL complexes occurs via autophagy. To analyze that, we used the specific autophagy inhibitor chloroquine (CQ) [26]. Under the conditions we selected, p62 and ATG8 were accumulated in the presence of CQ without any influence on cell viability (Figure S2). As shown in Figure 3A, CQ inhibits the degradation both of CUL4A and of selected CSN subunits, CSN3, CSN5, CSN7A, and CSN7B, in the same manner. Degradation in the presence of CSN5i-3 after 28 h incubation is about 50% and can be restored by CQ to almost 100% (Figure 3B). There is no influence of ATG7, an E1 activating enzyme of ATG8 conjugation, on CSN-CRL degradation (Figure S1A).
CSN-CRL degradation, through an autophagic mechanism, was further tested via fluorescence microscopy. The binding of CSN-CRL complexes to ATG8 was initially analyzed using confocal fluorescence microscopy [25]. The resulting images demonstrate a co-localization between CSN7A and ATG8. The process was inhibited by CQ (Figure 3C). CSN-CRL complexes might bind to ATG8 via specific autophagic receptors. Precipitates were tested to identify whether autophagic receptors bind prominent receptors, p62 and TOLLIP [27,28,29], to CSN or to CRL3. There was no association between the analyzed receptors and CSN or CRL3 (Figure S1B). RAB7 is a component that tethers complexes to autophagosomes [30] and plays a key role as a regulator of autophagosomes [26]. Therefore, whether the CSN-CRL complexes co-localize with RAB7 was also tested. The data shows that the marker of autophagosomes RAB7 is co-localized with CSN3, a component of CSN-CRL complexes (Figure 3D). Clearly, CSN3 is expelled from the nucleus and trapped by RAB7 vesicles in both LiSa-2 cells (Figure 3D) and HeLa cells (Figure S1C). Interestingly, in Figure 3D and Figure S1C, the vesicles, probably autophagosomes, are filled with complexes like CSN-CRL. In the presence of CQ, the autophagic process is stopped, and the superposition of CSN3 and RAB7 in the cytosol is even more evident in both LiSa-2 cells (Figure 3D) and HeLa cells (Figure S1C).

3.4. Neddylation-Dependent Self-Ubiquitylation of CUL3 and CUL4 Are Signals for CSN-CRL Degradation via Selective Macroautophagy

Endogenous immunoprecipitation using the anti-CSN7A antibody usually precipitates CSN subunits and cullins [1]. After 28 h of the incubation of LiSa-2 cells in the presence of 1 mM CSN5i-3, NEDD8 and ubiquitin labels were found in the molecular weight regions of the cullins (Figure 4A). Similarly, in FLAG-CSN7A pulldowns of LiSa-2 cells, NEDD8 and ubiquitin were detected using anti-NEDD8 and anti-ubiquitin antibodies in cullin positions (Figure 4B). To ensure that the cullins are ubiquitylated as a signal of the macroautophagy of CSN-CRL complexes, HeLa cells were stably transfected with HA-Ub. After 28 h of incubation without and with CSN5i-3, HA was immunoprecipitated. The results in Figure 4C show that, in the presence of CSN5i-3, the cullins and CSN subunits are immunoprecipitated as well as degraded, and that the cullins are labeled with ubiquitin. Moreover, in Figure S2A, the ubiquitylation of CUL4A is shown directly via immunoprecipitation under denaturing conditions. Clearly, cullin self-ubiquitylation is a signal for selective CSN-CRL macroautophagy. Is neddylation also necessary for autophagy? The neddylation inhibitor MLN4924 blocks the export of CSN-CRL complexes from the nucleus to the cytosol (Figure 4D). Moreover, it can be seen from Figure 4E that MLN4924 inhibits selective autophagy induced by CSN5i-3. Whereas 28 h of incubation with CSN5i-3 leads to the autophagy of about 50% of the CSN-CRL complexes, MLN4924 + CSN5i-3 or MLN4924 alone blocked the process almost completely (Figure 4F). It should be noted that the MLN4924 inhibitor must be added at least 2 h before the CSN5i-3 supplement. Adding MLN4924 at the same time as CSN5i-3 or later has no effect.

4. Discussion

Our results show that the complexes CSNCSN7A-CRL3 and CSNCSN7B-CRL4A are degraded after serum starvation via bulk autophagy or, in the presence of CSN5i-3, by selective macroautophagy (Figure 5). The data form the basis for a recent publication [17]. The self-ubiquitylation of CUL3 and CUL4, presumably mediated by the RBX protein of the appropriate CRLs, serves as a signal for selective macroautophagy (Figure 5). This was shown through stable HA-ubiquitin transfection and HA immunoprecipitation (Figure 4C). Generally, the special topology of ubiquitylation, such as its mono-ubiquitylation, drives protein complexes, organelles, and pathogens to autophagic degradation [31]. Most CSNCSN7A-CRL3 and CSNCSN7B-CRL4A complexes are localized in the nucleus. In CSN-CRL complexes, the binding of CSN5i-3 to the active site [23] of the deneddylation-active MPN + protein, CSN5, leads to the expulsion of the complexes from the nucleus (see Figure 2A and Figure 5). The autophagy machinery is localized in the cytoplasm. Interestingly, 26S proteasomes dissociate into subcomplexes prior to export and lysosomal degradation [19]. Regarding CSN-CRL complexes, it is not yet precisely known how stable non-functioning CSN-CRL particles are recognized and expelled from the nucleus. The degradation occurs via autophagy because the specific lysosome and autophagy inhibitor, CQ, blocks this process (Figure 3A) [32]. Furthermore, confocal fluorescence microscopy demonstrates that, in the presence of CSN5i-3, CSNCSN7A-CRL3 is most likely co-localized with ATG8 in the cytosol, where selective macroautophagy takes place (Figure 3C, upper images). CQ blocks this process (Figure 3C, lower images). Interestingly, the downregulation of ATG7, the E1-enzyme for ATGylation, does not significantly influence the autophagic degradation of CSN-CRL (Figure S1A), as confirmed by previously published proteaphagy data for mammalian cells [33]. The independence of CSN-CRL degradation from ATG7 could be explained by the occurrence of an as yet unknown further isoform of the E1 enzyme for ATGylation in Mammalia. The co-localization of CSN (CSN3) and RAB7 underlines the association of CSN particles with autophagosomes in HeLa cells (Figure S1C) [34]. In LiSa-2 cells (Figure 3D), as well as in HeLa cells (Figure S1C), confocal fluorescence imaging shows RAB7 vesicles, which might be filled with CSN particles. The loading of RAB7 vesicles is inhibited by CQ both in HeLa (Figure S1C) and in LiSa-2 cells (Figure 3D). Therefore, CSNCSN7A-CRL3 and CSNCSN7B-CRL4A are degraded via bulk autophagy and specific macroautophagy. We speculate that most CSN-CRL complexes are removed via autophagy. The selective macroautophagy of CSN-CRL complexes is similar to the proteaphagy of the 26S proteasome [19]. In both cases, for the selective macroautophagy of CSN-CRL complexes and of the 26S proteasome, specific ubiquitylation is required. In proteaphagy, three ubiquitin ligases act sequentially to promote nuclear export and autophagy [35]. In contrast, the degradation of CSN-CRL complexes via selective macroautophagy is initiated through the self-ubiquitylation of cullins by the corresponding CRLs. Generally, CRLs are involved in the autophagic degradation of different proteins via ubiquitylation in different organisms [36,37]. However, here we see the CSN-dependent self-ubiquitylation of CRLs, a novel self-regulation mechanism. The neddylation inhibitor MLN4924 blocks self-ubiquitylation. It inhibits two processes: first, the export from the nucleus (Figure 4D), and second, degradation in the cytosol via selective macroautophagy (Figure 4D,F).
As is the case with CSN-CRL, the degradation of the UPS regulator CSN via autophagy demonstrates further functional mutual regulation between two mayor proteolytic pathways, UPS and autophagy. CSN and CRL control autophagy [38,39]. At the same time, under special conditions, they are self-consumed via autophagy. Moreover, both pathways overlap via ubiquitylation and their component, the E3 ligases.

5. Conclusions

Our study reveals the degradation pathway of CSNCSN7A-CRL3 and CSNCSN7B-CRL4 particles via bulk autophagy or, in the presence of CSN5i-3, via macroautophagy. CSN-CRL complexes signal macroautophagy through self-ubiquitylation, which can be blocked via neddylation inhibition. Ubiquitylated CSN-CRL complexes are tethered by, thus far, unknown autophagy receptors (ARs) and ATG8 to phagophores and are, finally, degraded in lysosomes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom16020218/s1, Figure S1: No influence of ATG7 and of specific autophagic receptors, p62 and TOLLIP, and effect of chloroquine on the degradation of CSN-CRL complexes by macroautophagy; Figure S2: Direct ubiquitylation of CUL4A and influence of CSN5i-3 and CQ on autophagic markers; Figure S3: Original images of Western blots shown in Figure 1. Figure S4: Original images of Western blots shown in Figure 2. Figure S5: Original images of Western blots shown in Figure 3. Figure S6: Original images of Western blots shown in Figure 4. Figure S7: Original images of Western blots shown in Figures S1 and S2.

Author Contributions

D.D., R.H. and W.D. designed the research. D.D. and R.H. performed the experiments and analyzed the data. W.D., D.D. and R.H. supervised. W.D. and D.D. conducted the statistical analysis. W.D. and D.D. wrote the manuscript. W.D. was responsible for funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by a grant of the European Union Program European Regional Development Fund of the Ministry of Economy, Science and Digitalisation in Saxony Anhalt within the Centre of Dynamic Systems (ZS/2016/04/78155). We acknowledge support from the Open Access Publication fund of the Medical Faculty of Otto-von-Guericke-University Magdeburg.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

We thank E. Mark, P. Wendler and J. Wenzel for technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ATG8Autophagy-related protein 8
CAND1Cullin-associated and neddylation-dissociated 1
CSNCOP9 signalosome
CRLCullin-RING ubiquitin ligase
CQChloroquine
DUBDeubiquitylating enzyme
SRSubstrate receptor
UPSUbiquitin proteasome system

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Figure 1. Degradation of COP9 signalosome after serum starvation and specific inhibition via CSN5i-3. (A) Incubation of LiSa-2 cells for 28 h and 48 h, with and without serum, and in the absence and in the presence of CSN5i-3 at 37 °C. The deneddylation inhibitor CSN5i-3 was used at a concentration of 1 µM. The 26S proteasome subunits RPN1 and 20Sa4 served as controls. After incubation, the cells were lyzed with triple lysis buffer and blotted. Blots were analyzed with anti-CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, -RPN1, and -20Sa4 antibodies. The anti-γ-tubulin antibody served as a loading control. (B) Blots of LiSa-2 cells shown in (A) were quantified via densitometry to determine the relative amounts of CSN3, CSN5, CSN7A, and CSN7B with and without serum, as well as without and with 1 µM CSN5i-3 after 0 h, 28 h, and 48 h. Relative amounts at time 0 h were set to 100%, and relative amounts at 28 h and 48 h were related to values at 100%. Data is expressed as relative amounts of CSN3, CSN5, CSN7A, and CSN7B at 28 h or 48 h related to relative amounts at 0 h plus/minus standard deviations (SDs, n = 3). (C) HeLa–wildtype (HeLa-WT), HeLa-CSN7A-KO (CSN7A-KO), and HeLa-CSN7B-KO (CSN7B-KO) cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After incubation, the cells were lyzed with triple lysis buffer, and blots were analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. The anti-γ-tubulin antibody served as a loading control. (D) LiSa-2 cells with FLAG-vector (FLAG), LiSa-2-FLAG-CSN7A (FLAG-CSN7A), and LiSa-2-FLAG-CSN7B (FLAG-CSN7B) were incubated without and with 0.5 µM and 1 µM CSN5i-3 inhibitor at 37 °C. Upon incubation, the cells were lyzed with triple lysis buffer, and blots were analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, and -CSN7B antibodies. FLAG was used as a control. The anti-γ-tubulin antibody served as a loading control. (E) HeLa, B8, NCI, AGS, and LiSa-2 cells were incubated for 28 h without and with 1 µM CSN5i-3 at 37 °C. After lysis, blots were analyzed with anti-CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. The anti-γ-tubulin antibody served as a loading control. The original Western blot images are shown in Supplementary Materials Figure S3.
Figure 1. Degradation of COP9 signalosome after serum starvation and specific inhibition via CSN5i-3. (A) Incubation of LiSa-2 cells for 28 h and 48 h, with and without serum, and in the absence and in the presence of CSN5i-3 at 37 °C. The deneddylation inhibitor CSN5i-3 was used at a concentration of 1 µM. The 26S proteasome subunits RPN1 and 20Sa4 served as controls. After incubation, the cells were lyzed with triple lysis buffer and blotted. Blots were analyzed with anti-CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, -RPN1, and -20Sa4 antibodies. The anti-γ-tubulin antibody served as a loading control. (B) Blots of LiSa-2 cells shown in (A) were quantified via densitometry to determine the relative amounts of CSN3, CSN5, CSN7A, and CSN7B with and without serum, as well as without and with 1 µM CSN5i-3 after 0 h, 28 h, and 48 h. Relative amounts at time 0 h were set to 100%, and relative amounts at 28 h and 48 h were related to values at 100%. Data is expressed as relative amounts of CSN3, CSN5, CSN7A, and CSN7B at 28 h or 48 h related to relative amounts at 0 h plus/minus standard deviations (SDs, n = 3). (C) HeLa–wildtype (HeLa-WT), HeLa-CSN7A-KO (CSN7A-KO), and HeLa-CSN7B-KO (CSN7B-KO) cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After incubation, the cells were lyzed with triple lysis buffer, and blots were analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. The anti-γ-tubulin antibody served as a loading control. (D) LiSa-2 cells with FLAG-vector (FLAG), LiSa-2-FLAG-CSN7A (FLAG-CSN7A), and LiSa-2-FLAG-CSN7B (FLAG-CSN7B) were incubated without and with 0.5 µM and 1 µM CSN5i-3 inhibitor at 37 °C. Upon incubation, the cells were lyzed with triple lysis buffer, and blots were analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, and -CSN7B antibodies. FLAG was used as a control. The anti-γ-tubulin antibody served as a loading control. (E) HeLa, B8, NCI, AGS, and LiSa-2 cells were incubated for 28 h without and with 1 µM CSN5i-3 at 37 °C. After lysis, blots were analyzed with anti-CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. The anti-γ-tubulin antibody served as a loading control. The original Western blot images are shown in Supplementary Materials Figure S3.
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Figure 2. COP9 signalosome and cullin-RING ubiquitin ligases are degraded as an entire complex. (A) HeLa and LiSa-2 cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After 28 h, the cells were fractionated into cytosol (C), nucleus 1 (N1), and nucleus 2 (N2) (see Materials and Methods). The fractions were blotted and analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. Anti-GAPDH served as a control for the cytosol fraction; for the N1 fraction, anti-C23 was the control, and for N2, it was anti-LAMIN B2 antibodies. (B) LiSa-2 cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After the incubation, the cells were lyzed with mono-lysis buffer, and lysates were immunoprecipitated with anti-CSN3 and anti-CUL3 antibodies. The LiSa-2 cell lysate (input) and immunoprecipitates obtained with anti-CSN3 and anti-CUL3 antibodies were blotted and analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, and anti-CSN7B antibodies. The anti-γ-tubulin antibody served as a loading control. (C) In immunoprecipitates, relative amounts of CUL4A, CSN3, and CSN7B were determined via densitometry using the anti-CSN3 antibody, as shown in (B). The columns represent ratios between relative amounts of CUL4A divided by relative amounts of the CSN subunits CSN3 (left panel) or CSN7B (right panel), in the absence (control) and presence of CSN5i-3, plus/minus standard deviations (SDs, n = 3). (D) HeLa cells were incubated at 37 °C without or with 1 µM CSN5i-3. After 28 h, the cells were lyzed with mono-lysis buffer (input) and lysate was loaded onto a 10% to 30% glycerol gradient (see Materials and Methods). After density gradient centrifugation, the fractions were analyzed using the appropriate antibodies. CDC48 was used as a control. The original Western blot images are shown in Supplementary Materials Figure S4. (E) LiSa-2 cells were analyzed via confocal fluorescence microscopy. After 28 h of incubation at 37 °C, in the absence (control) and in the presence of 1 µM CSN5i-3, the cells were fixed and stained with specific antibodies, including CSN7A (red) and LAMP2 (green), and analyzed via confocal microscopy. Cell nuclei were stained with DAPI (blue). The yellow bar in the “Composite” channel corresponds to 10 µm.
Figure 2. COP9 signalosome and cullin-RING ubiquitin ligases are degraded as an entire complex. (A) HeLa and LiSa-2 cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After 28 h, the cells were fractionated into cytosol (C), nucleus 1 (N1), and nucleus 2 (N2) (see Materials and Methods). The fractions were blotted and analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. Anti-GAPDH served as a control for the cytosol fraction; for the N1 fraction, anti-C23 was the control, and for N2, it was anti-LAMIN B2 antibodies. (B) LiSa-2 cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After the incubation, the cells were lyzed with mono-lysis buffer, and lysates were immunoprecipitated with anti-CSN3 and anti-CUL3 antibodies. The LiSa-2 cell lysate (input) and immunoprecipitates obtained with anti-CSN3 and anti-CUL3 antibodies were blotted and analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, and anti-CSN7B antibodies. The anti-γ-tubulin antibody served as a loading control. (C) In immunoprecipitates, relative amounts of CUL4A, CSN3, and CSN7B were determined via densitometry using the anti-CSN3 antibody, as shown in (B). The columns represent ratios between relative amounts of CUL4A divided by relative amounts of the CSN subunits CSN3 (left panel) or CSN7B (right panel), in the absence (control) and presence of CSN5i-3, plus/minus standard deviations (SDs, n = 3). (D) HeLa cells were incubated at 37 °C without or with 1 µM CSN5i-3. After 28 h, the cells were lyzed with mono-lysis buffer (input) and lysate was loaded onto a 10% to 30% glycerol gradient (see Materials and Methods). After density gradient centrifugation, the fractions were analyzed using the appropriate antibodies. CDC48 was used as a control. The original Western blot images are shown in Supplementary Materials Figure S4. (E) LiSa-2 cells were analyzed via confocal fluorescence microscopy. After 28 h of incubation at 37 °C, in the absence (control) and in the presence of 1 µM CSN5i-3, the cells were fixed and stained with specific antibodies, including CSN7A (red) and LAMP2 (green), and analyzed via confocal microscopy. Cell nuclei were stained with DAPI (blue). The yellow bar in the “Composite” channel corresponds to 10 µm.
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Figure 3. COP9 signalosome–cullin-RING ubiquitin ligase complexes are degraded via selective macroautophagy. (A) HeLa cells were incubated for 28 h at 37 °C in the absence (control) and the presence of 1 µM CSN5i-3 or with 1 µM CSN5i-3, and with 30 µM of the inhibitor chloroquine (CQ). Original images of Western blots are shown in Supplementary Materials Figure S5. (B) In the blots, as in (A), relative amounts of CUL4A, CSN3, CSN5, CSN7A, and CSN7B were determined via densitometry in HeLa cells. The columns represent relative amounts of CUL4A, CSN3, CSN5, CSN7A, and CSN7B without (control) and with 1 µM CSN5i-3 or with both 1 µM CSN5i-3 and 30 µM of chloroquine (CQ) plus/minus standard deviations (SDs, n = 3). (C) HeLa cells were analyzed via confocal fluorescence microscopy. After 28 h of incubation at 37 °C in the presence of 1 µM CSN5i-3, the cells were fixed and stained with specific antibodies, including CSN7A (red) and ATG8 (green), and analyzed via confocal microscopy (upper panel). In the lower panel, the autophagy-specific inhibitor chloroquine (30 µM) was added, which inhibited the process and made the images analyzed via confocal microscopy more visible. Cell nuclei were stained with DAPI (blue). The yellow bar in the “Composite” channel corresponds to 10 µm. (D) LiSa-2 cells were analyzed via confocal fluorescence microscopy. After 28 h of incubation at 37 °C in the presence of 1 µM CSN5i-3, the cells were fixed and stained with specific antibodies, including CSN3 (green) and RAB7 (red), and analyzed via confocal microscopy (upper panel). In the lower panel, the autophagy-specific inhibitor CQ (30 µM) was added, which inhibited the process and made the imaging more visible. Cell nuclei were stained with DAPI (blue). The yellow bar in the “Composite” channel corresponds to 10 µm.
Figure 3. COP9 signalosome–cullin-RING ubiquitin ligase complexes are degraded via selective macroautophagy. (A) HeLa cells were incubated for 28 h at 37 °C in the absence (control) and the presence of 1 µM CSN5i-3 or with 1 µM CSN5i-3, and with 30 µM of the inhibitor chloroquine (CQ). Original images of Western blots are shown in Supplementary Materials Figure S5. (B) In the blots, as in (A), relative amounts of CUL4A, CSN3, CSN5, CSN7A, and CSN7B were determined via densitometry in HeLa cells. The columns represent relative amounts of CUL4A, CSN3, CSN5, CSN7A, and CSN7B without (control) and with 1 µM CSN5i-3 or with both 1 µM CSN5i-3 and 30 µM of chloroquine (CQ) plus/minus standard deviations (SDs, n = 3). (C) HeLa cells were analyzed via confocal fluorescence microscopy. After 28 h of incubation at 37 °C in the presence of 1 µM CSN5i-3, the cells were fixed and stained with specific antibodies, including CSN7A (red) and ATG8 (green), and analyzed via confocal microscopy (upper panel). In the lower panel, the autophagy-specific inhibitor chloroquine (30 µM) was added, which inhibited the process and made the images analyzed via confocal microscopy more visible. Cell nuclei were stained with DAPI (blue). The yellow bar in the “Composite” channel corresponds to 10 µm. (D) LiSa-2 cells were analyzed via confocal fluorescence microscopy. After 28 h of incubation at 37 °C in the presence of 1 µM CSN5i-3, the cells were fixed and stained with specific antibodies, including CSN3 (green) and RAB7 (red), and analyzed via confocal microscopy (upper panel). In the lower panel, the autophagy-specific inhibitor CQ (30 µM) was added, which inhibited the process and made the imaging more visible. Cell nuclei were stained with DAPI (blue). The yellow bar in the “Composite” channel corresponds to 10 µm.
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Figure 4. Ubiquitylation of CUL3 and CUL4 is dependent on neddylation for selective CSN-CRL macroautophagy. (A) LiSa-2 cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After incubation, the cells were lyzed with mono-lysis buffer, and the lysates were immunoprecipitated with the anti-CSN7A antibody. LiSa-2 cell lysate (input) and the immunoprecipitates obtained with the anti-CSN7A antibody were botted and analyzed with anti-CUL3, -CUL4A, -Ub, -NEDD8, -CSN3, -CSN5, and -CSN7A antibodies. The anti-γ-tubulin antibody served as a loading control. (B) LiSa-2 cells stably transfected with FLAG-CSN7A were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After incubation, the cells were lyzed with mono-lysis buffer, and the lysates were pulled down with FLAG-CSN7A. LiSa-2-FLAG-CSN7A cell lysate (input) and the pulldowns obtained with FLAG-CSN7A were botted and analyzed with anti-CUL3, -CUL4A, -Ub, -NEDD8, -CSN3, -CSN5, -CSN7A, and -CSN7B antibodies. FLAG served as a control for the input and in FLAG-CSN7A pulldown. The anti-γ-tubulin antibody served as a loading control. (C) HeLa cells stably transfected with HA-ubiquitin (HA-Ub) were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. Upon incubation, the cells were lyzed with mono-lysis buffer, and the lysates were immunoprecipitated by HA. HeLa cell lysate with stably transfected HA-Ub (input) and immunoprecipitated with the anti-HA antibody were blotted and analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN7A, and -CSN7B antibodies. HeLa cells without stably transfected HA-Ub served as a control. The anti-γ-tubulin antibody served as a loading control. (D) HeLa cells were incubated without and with 1 µM CSN5i-3, with 1 µM MLN4924 (pretreatment for 2 h), or with both 1 µM CSN5i-3 and 1 µM MLN4924 (pretreatment for 2 h) for 28 h at 37 °C. After 28 h, the cells were fractionated into cytosol (C), nucleus 1 (N1), and nucleus 2 (N2) (see Materials and Methods). The fractions were blotted and analyzed using anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. Anti-GAPDH served as a control for the cytosol fraction, anti-C23 served as a control for the N1 fraction, and anti-LAMIN B2 antibodies served as a control for N2. (E) LiSa-2 cells were incubated without and with 1 µM CSN5i-3, with both 1 µM CSN5i-3 and 1 µM MLN4924 (pretreatment for 2 h), or with 1 µM MLN4924 (pretreatment for 2 h) for 28 h at 37 °C. After incubation, the cells were lyzed with triple lysis buffer. LiSa-2 cell lysates were botted and analyzed with anti-CUL3, -CUL4A, -NEDD8, -CSN3, -CSN5, -CSN7A, and -CSN7B antibodies. The anti-γ-tubulin antibody served as a loading control. The original Western blot images are shown in Supplementary Materials Figure S6. (F) In the blots, as in (E), the relative amounts of CUL3, CUL4A, CSN3, CSN5, CSN7A, and CSN7B were determined via densitometry in LiSa-2 cells. The columns represent relative amounts of CUL3, CUL4A, CSN3, CSN5, CSN7A, and CSN7B without (control) and with 1 µM CSN5i-3, with both 1 µM CSN5i-3 and 1 µM MLN4924, and with 1 µM MLN4924 plus/minus standard deviations (SDs, n = 3).
Figure 4. Ubiquitylation of CUL3 and CUL4 is dependent on neddylation for selective CSN-CRL macroautophagy. (A) LiSa-2 cells were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After incubation, the cells were lyzed with mono-lysis buffer, and the lysates were immunoprecipitated with the anti-CSN7A antibody. LiSa-2 cell lysate (input) and the immunoprecipitates obtained with the anti-CSN7A antibody were botted and analyzed with anti-CUL3, -CUL4A, -Ub, -NEDD8, -CSN3, -CSN5, and -CSN7A antibodies. The anti-γ-tubulin antibody served as a loading control. (B) LiSa-2 cells stably transfected with FLAG-CSN7A were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. After incubation, the cells were lyzed with mono-lysis buffer, and the lysates were pulled down with FLAG-CSN7A. LiSa-2-FLAG-CSN7A cell lysate (input) and the pulldowns obtained with FLAG-CSN7A were botted and analyzed with anti-CUL3, -CUL4A, -Ub, -NEDD8, -CSN3, -CSN5, -CSN7A, and -CSN7B antibodies. FLAG served as a control for the input and in FLAG-CSN7A pulldown. The anti-γ-tubulin antibody served as a loading control. (C) HeLa cells stably transfected with HA-ubiquitin (HA-Ub) were incubated without and with 1 µM CSN5i-3 for 28 h at 37 °C. Upon incubation, the cells were lyzed with mono-lysis buffer, and the lysates were immunoprecipitated by HA. HeLa cell lysate with stably transfected HA-Ub (input) and immunoprecipitated with the anti-HA antibody were blotted and analyzed with anti-CUL3, -CUL4A, -CSN3, -CSN7A, and -CSN7B antibodies. HeLa cells without stably transfected HA-Ub served as a control. The anti-γ-tubulin antibody served as a loading control. (D) HeLa cells were incubated without and with 1 µM CSN5i-3, with 1 µM MLN4924 (pretreatment for 2 h), or with both 1 µM CSN5i-3 and 1 µM MLN4924 (pretreatment for 2 h) for 28 h at 37 °C. After 28 h, the cells were fractionated into cytosol (C), nucleus 1 (N1), and nucleus 2 (N2) (see Materials and Methods). The fractions were blotted and analyzed using anti-CUL3, -CUL4A, -CSN3, -CSN5, -CSN7A, -CSN7B, and -CSN8 antibodies. Anti-GAPDH served as a control for the cytosol fraction, anti-C23 served as a control for the N1 fraction, and anti-LAMIN B2 antibodies served as a control for N2. (E) LiSa-2 cells were incubated without and with 1 µM CSN5i-3, with both 1 µM CSN5i-3 and 1 µM MLN4924 (pretreatment for 2 h), or with 1 µM MLN4924 (pretreatment for 2 h) for 28 h at 37 °C. After incubation, the cells were lyzed with triple lysis buffer. LiSa-2 cell lysates were botted and analyzed with anti-CUL3, -CUL4A, -NEDD8, -CSN3, -CSN5, -CSN7A, and -CSN7B antibodies. The anti-γ-tubulin antibody served as a loading control. The original Western blot images are shown in Supplementary Materials Figure S6. (F) In the blots, as in (E), the relative amounts of CUL3, CUL4A, CSN3, CSN5, CSN7A, and CSN7B were determined via densitometry in LiSa-2 cells. The columns represent relative amounts of CUL3, CUL4A, CSN3, CSN5, CSN7A, and CSN7B without (control) and with 1 µM CSN5i-3, with both 1 µM CSN5i-3 and 1 µM MLN4924, and with 1 µM MLN4924 plus/minus standard deviations (SDs, n = 3).
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Figure 5. Model of the selective macroautophagic degradation of CSN-CRL complexes in the cytoplasm and in the nucleus. The selective macroautophagy of CSN-CRL complexes in the presence of the specific deneddylation inhibitor, CSN5i-3, is demonstrated in the model. As a trigger for autophagy, CSN-CRL complexes cause the self-ubiquitylation of cullins, which is initiated by CSN5i-3 in the cytoplasm and in the nucleus. In the presence of the inhibitor, the neddylated and self-ubiquitylated complexes leave the nucleus. The subsequent selective macroautophagy steps take place in the cytoplasm. CSN-CRL complexes are tethered via autophagy receptors (ARs) and ATG8 to phagophores. The phagophores transform into autophagosomes, which merge with the help of RAB7 with lysosomes.
Figure 5. Model of the selective macroautophagic degradation of CSN-CRL complexes in the cytoplasm and in the nucleus. The selective macroautophagy of CSN-CRL complexes in the presence of the specific deneddylation inhibitor, CSN5i-3, is demonstrated in the model. As a trigger for autophagy, CSN-CRL complexes cause the self-ubiquitylation of cullins, which is initiated by CSN5i-3 in the cytoplasm and in the nucleus. In the presence of the inhibitor, the neddylated and self-ubiquitylated complexes leave the nucleus. The subsequent selective macroautophagy steps take place in the cytoplasm. CSN-CRL complexes are tethered via autophagy receptors (ARs) and ATG8 to phagophores. The phagophores transform into autophagosomes, which merge with the help of RAB7 with lysosomes.
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Dubiel, D.; Hartig, R.; Dubiel, W. The Degradation Pathway of COP9 Signalosome–Cullin-RING Ubiquitin Ligase Complexes via Autophagy. Biomolecules 2026, 16, 218. https://doi.org/10.3390/biom16020218

AMA Style

Dubiel D, Hartig R, Dubiel W. The Degradation Pathway of COP9 Signalosome–Cullin-RING Ubiquitin Ligase Complexes via Autophagy. Biomolecules. 2026; 16(2):218. https://doi.org/10.3390/biom16020218

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Dubiel, Dawadschargal, Roland Hartig, and Wolfgang Dubiel. 2026. "The Degradation Pathway of COP9 Signalosome–Cullin-RING Ubiquitin Ligase Complexes via Autophagy" Biomolecules 16, no. 2: 218. https://doi.org/10.3390/biom16020218

APA Style

Dubiel, D., Hartig, R., & Dubiel, W. (2026). The Degradation Pathway of COP9 Signalosome–Cullin-RING Ubiquitin Ligase Complexes via Autophagy. Biomolecules, 16(2), 218. https://doi.org/10.3390/biom16020218

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