Ub-like Proteins Research Articles

Abstract 30: Nub1l Suppresses Neddylation And Enhances Misfolded Protein Clearance In Cardiomyocytes

Protein modification by ubiquitin (Ub) or Ub-like proteins such as NEDD8 (neddylation) constitutes a fundamental regulatory mechanism of protein function. In contrast to well-recognized role of Ub in protein degradation, little is known about the role of NEDD8 in protein quality control. We have previously revealed that CM-restricted inactivation of deneddylation, a process that removes NEDD8 from modified proteins, accumulates neddylated proteins and impairs proteasomal and autophagic proteolysis. Here we report that proteasome inhibitors, simulated ischemia/reperfusion and H2O2 significantly increase NEDD8 conjugates in cardiomyocytes (CMs). Immunoprecipitation analysis reveals mixed modification of these proteins by Ub and NEDD8. Expression of NEDD8 but not the conjugation-deficient mutant increases neddylated proteins and accumulates a proteasome surrogate substrate GFPu in a dose-dependent manner, suggesting that excessive neddylation disrupts proteasomal proteolysis. We further targets to NUB1L, a UBL (Ub-like domain)-UBA (Ub associating domain) family protein that was shown to negatively regulate neddylation. NUB1L expression markedly reduces free NEDD8 by promoting its degradation, and abrogates proteasome inhibition-induced neddylation in CMs. Suppression of neddylation by NUB1L expression enhances GFPu degradation at baseline, and attenuates GFPu accumulation upon sI/R and H2O2 treatment. Furthermore, NUB1L expression promotes, while down-regulation of NUB1L impairs, the clearance of a bona fide misfolded protein in CMs. NUB1L expression also ameliorates proteotoxic stress- and sI/R-induced CM injury. Finally, increased NEDD8 conjugates are evident in the mouse hearts of a number of cardiac disease models as well as in human failing hearts. Together, our findings suggest that excessive neddylation disrupts protein quality control and that antagonizing neddylation by NUB1L promotes misfolded protein degradation. Targeting neddylation/NUB1L could be a novel therapeutic strategy for prevention and treatment of insufficient protein quality control-associated cardiac disease.

Prokaryotic ubiquitin-like protein modification.

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers.

Nonenzymatic rubylation and ubiquitination of proteins for structural and functional studies.

Uncovering the mechanisms that allow conjugates of ubiquitin (Ub) and/or Ub-like (UBL) proteins such as Rub1 to serve as distinct molecular signals requires the ability to make them with native connectivity and defined length and linkage composition. A novel, effective, and affordable strategy for controlled chemical assembly of fully natural UBL-Ub, Ub-UBL, and UBL-UBL conjugates from recombinant monomers is presented. Rubylation of Ub and Rub1 and ubiquitination of Rub1 was achieved without E2/E3 enzymes. New residue-specific information was obtained on the interdomain contacts in naturally-occurring K48-linked Rub1-Ub and Ub-Rub1, and K29-linked Rub1-Ub heterodimers, and their recognition by a K48-linkage-specific Ub receptor. The disassembly of these heterodimers by major deubiquitinating enzymes was examined and it was discovered that some deubiquitinases also possess derubylase activity. This unexpected result suggests possible crosstalk between Ub and Rub1/Nedd8 signaling pathways.

Examination of ubiquitination and SUMOlyation of GRK4γ (1055.3)

G protein‐coupled receptor kinases 4γ (GRK4γ) is associated with hypertension. However, the molecular mechanism of action of GRK4γ remains largely unknown. From a proteomic examination of the binding partners of GRK4γ, we identified Gαs, Heat Shock Protein 90 (Hsp90) and an E3 ubiquitin ligase as potential binding partners. Additionally, previous studies suggested that GRK4γ is ubiquitinated. GRK2 and GRK5 are known to be ubiquitinated while SUMOylation, a small Ub‐like modifier protein that also attaches to lysine, has not been extensively studied in any GRKs. Therefore, we tested the hypothesis that GRK4γ can be ubiquitinated and SUMOylated. To demonstrate Ubiquitination and SUMOylation, we immunoprecipitated (IPed) GRK4γ and blotted for Ub or SUMO. The data clearly showed that GRK4γ is ubiquitinated but not SUMOylated. Interesting, GRK4γ ubiquitination is significantly enhanced after stimulation of MG132, a cell‐permeable proteasome inhibitor, but remains unchanged in the presence of two other proteasome inhibitors (Lactacystin and proteasome inhibitor I) which is counter to the previous studies. Further studies will be conducted to identify the role of GRK4γ activity in relation to GRK4γ ubiquination as well as identify the ubiquitination sites on GRK4γ and the role that ubiquitination plays in GRK4γ activity.

An improved workflow for identifying ubiquitin/ubiquitin-like protein conjugation sites from tandem mass spectra

The identification of ubiquitin (Ub) and Ub-like protein (Ubl) conjugation sites is important in understanding their roles in biological pathway regulations. However, unambiguously and sensitively identifying Ub/Ubl conjugation sites through high-throughput MS remains challenging. We introduce an improved workflow for identifying Ub/Ubl conjugation sites based on the ChopNSpice and X!Tandem software. ChopNSpice is modified to generate Ub/Ubl conjugation peptides in the form of a cross-link. A combinatorial FASTA database can be acquired using the modified ChopNSpice (MchopNSpice). The modified X!Tandem (UblSearch) introduces a new fragmentation model for the Ub/Ubl conjugation peptides to match unambiguously the MS/MS spectra with linear peptides or Ub/Ubl conjugation peptides using the combinatorial FASTA database. The novel workflow exhibited better performance in analyzing an Ub and Ubl spectral library and a large-scale Trypanosoma cruzi small Ub-related modifier dataset compared with the original ChopNSpice method. The proposed workflow is more suitable for processing large-scale MS datasets of Ub/Ubl modification. MchopNSpice and UblSearch are freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/maublsearch.

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB∼NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a “gate-keeping” role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.

ChemInform Abstract: The Lysine48‐Based Polyubiquitin Chain Proteasomal Signal: Not a Single Child Anymore

AbstractReview: ca. 70 refs.

Diversity of Ubiquitin and ISG15 Specificity among Nairoviruses' Viral Ovarian Tumor Domain Proteases

Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferon-stimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub- and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.

Ubiquitination in plant nutrient utilization

Ubiquitin (Ub) is well-established as a major modifier of signaling in eukaryotes. However, the extent to which plants rely on Ub for regulating nutrient uptake is still in its infancy. The main characteristic of ubiquitination is the conjugation of Ub onto lysine residues of acceptor proteins. In most cases the targeted protein is rapidly degraded by the 26S proteasome, the major proteolysis machinery in eukaryotic cells. The Ub-proteasome system is responsible for removing most abnormal peptides and short-lived cellular regulators, which, in turn, control many processes. This allows cells to respond rapidly to intracellular signals and changing environmental conditions. This perspective will discuss how plants utilize Ub conjugation for sensing environmental nutrient levels. We will highlight recent advances in understanding how Ub aids nutrient homeostasis by affecting the trafficking of membrane bound transporters. Given the overrepresentation of genes encoding Ub-metabolizing enzymes in plants, intracellular signaling events regulated by Ub that lead to transcriptional responses due to nutrient starvation is an under explored area ripe for new discoveries. We provide new insight into how Ub based biochemical tools can be exploited to reveal new molecular components that affect nutrient signaling. The mechanistic nature of Ub signaling indicates that dominant form of any new molecular components can be readily generated and are likely shed new light on how plants cope with nutrient limiting conditions. Finally as part of future challenges in this research area we introduce the newly discovered roles of Ub-like proteins in nutrient homeostasis.

The Lysine48‐Based Polyubiquitin Chain Proteasomal Signal: Not a Single Child Anymore

The conjugation of ubiquitin (Ub) to proteins is involved in the regulation of many processes. The modification serves as a recognition element in trans, in which downstream effectors bind to the modified protein and determine its fate and/or function. A polyUb chain that is linked through internal lysine (Lys)-48 of Ub and anchored to an internal Lys residue of the substrate has become the accepted "canonical" signal for proteasomal targeting and degradation. However, recent studies show that the signal is far more diverse and that chains based on other internal linkages, as well as linear or heterologous chains made of Ub and Ub-like proteins and even monoUb, are recognized by the proteasome. In addition, chains linked to residues other than internal Lys were described, all challenging the current paradigm.

Structural Aspects of Ubiquitin Binding Specificities

Ubiquitin (Ub) is widely distributed in eukaryotic cells as its name means. There are many kinds of Ub-like proteins (for example, SUMO, NEDD8 and ISG15) and Ub-like domains (UbLs) included in multi-domain proteins. To date, a large number of Ub-binding domains (UBDs), such as UBA, CUE, UIM, ZnF, and Pru, are coming up to us with different affinities to Ub and its homologues. The binding specificities provide the basis for controlling various cellular events as well as for delivering ubiquitinated proteins to proteasome for degradation. Structural details of these UBDs and their complexes with Ub might as well show us the delicate mechanism of Ub recognition and regulation. This review summarizes recent progresses on deciphering the structure-based Ub-binding specificities, which are the importantly fundamental elements in orchestrating the ubiquitination and deubiquitination processes in eukaryotic cells.

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.

The natural history of ubiquitin and ubiquitin-related domains

The ubiquitin (Ub) system is centered on conjugation and deconjugation of Ub and Ub-like (Ubls) proteins by a system of ligases and peptidases, respectively. Ub/Ubls contain the beta-grasp fold, also found in numerous proteins with biochemically distinct roles unrelated to the conventional Ub-system. The beta-GF underwent an early radiation spawning at least seven clades prior to the divergence of extant organisms from their last universal common ancestor, first emerging in the context of translation-related RNA-interactions and subsequently exploding to occupy various functional niches. Most beta-GF diversification occurred in prokaryotes, with the Ubl clade showing dramatic expansion in the eukaryotes. Diversification of Ubl families in eukaryotes played a major role in emergence of characteristic eukaryotic cellular sub-structures and systems. Recent comparative genomics studies indicate precursors of the eukaryotic Ub-system emerged in prokaryotes. The simplest of these combine an Ubl and an E1-like enzyme in metabolic pathways. Sampylation in archaea and Urmylation in eukaryotes appear to represent recruitment of such systems as simple protein-tagging apparatuses. However, other prokaryotic systems incorporated further components and mirror the eukaryotic condition in possessing an E2, a RING-type E3 or both of these components. Additionally, prokaryotes have evolved conjugation systems independent of Ub ligases, such as the Pup system.

In Vivo RNAi Screen Reveals Neddylation Genes as Novel Regulators of Hedgehog Signaling

Hedgehog (Hh) signaling is highly conserved in all metazoan animals and plays critical roles in many developmental processes. Dysregulation of the Hh signaling cascade has been implicated in many diseases, including cancer. Although key components of the Hh pathway have been identified, significant gaps remain in our understanding of the regulation of individual Hh signaling molecules. Here, we report the identification of novel regulators of the Hh pathway, obtained from an in vivo RNA interference (RNAi) screen in Drosophila. By selectively targeting critical genes functioning in post-translational modification systems utilizing ubiquitin (Ub) and Ub-like proteins, we identify two novel genes (dUba3 and dUbc12) that negatively regulate Hh signaling activity. We provide in vivo and in vitro evidence illustrating that dUba3 and dUbc12 are essential components of the neddylation pathway; they function in an enzyme cascade to conjugate the ubiquitin-like NEDD8 modifier to Cullin proteins. Neddylation activates the Cullin-containing ubiquitin ligase complex, which in turn promotes the degradation of Cubitus interruptus (Ci), the downstream transcription factor of the Hh pathway. Our study reveals a conserved molecular mechanism of the neddylation pathway in Drosophila and sheds light on the complex post-translational regulations in Hh signaling.

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

The ubiquitin (Ub)-related modifier Urm1 functions as a sulfur carrier in tRNA thiolation by means of a mechanism that requires the formation of a thiocarboxylate at the C-terminal glycine residue of Urm1. However, whether Urm1 plays an additional role as a Ub-like protein modifier remains unclear. Here, we show that Urm1 is conjugated to lysine residues of target proteins and that oxidative stress enhances protein urmylation in both Saccharomyces cerevisiae and mammalian cells. Similar to ubiquitylation, urmylation involves a thioester intermediate and results in the formation of a covalent peptide bond between Urm1 and its substrates. In contrast to modification by canonical Ub-like modifiers, however, conjugation of Urm1 involves a C-terminal thiocarboxylate of the modifier. We have confirmed that the peroxiredoxin Ahp1 is such a substrate in S. cerevisiae and found that Urm1 targets a specific lysine residue of Ahp1 in vivo. In addition, we have identified several unique substrates in mammalian cells and show that Urm1 targets at least two pathways on oxidant treatment. First, Urm1 is appended to lysine residues of three components that function in its own pathway (i.e., MOCS3, ATPBD3, and CTU2). Second, Urm1 is conjugated to the nucleocytoplasmic shuttling factor cellular apoptosis susceptibility protein. Thus, Urm1 has a conserved dual role by integrating the functions of prokaryotic sulfur carriers with those of eukaryotic protein modifiers of the Ub family.

Systematic In Vivo RNAi Analysis Identifies IAPs as NEDD8-E3 Ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.

Therapeutically targeting the SUMOylation, Ubiquitination and Proteasome pathways as a novel anticancer strategy

The ubiquitin (Ub)+proteasome proteolytic pathway is responsible for the selective degradation of the majority of nuclear and cytosolic proteins. The proteasome is a high molecular weight multicatalytic protease that serves as the catalytic core of the complex Ub-dependent protein degradation pathway and is an exciting new target for the development of novel anticancer therapies. Inhibition of the proteasome, and consequently Ub-dependent proteolysis, with the small molecule pharmacologic agent bortezomib led to approval by the US Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) that has subsequently been extended to other hematologic malignancies. Inhibition of the proteasome results in the intracellular accumulation of many ubiquitinated proteins that control essential cellular functions such as cellular growth and apoptosis. The accumulation of high molecular weight Ub~protein conjugates eventually triggers apoptosis, with tumor cells more susceptible to proteasome inhibition than non-malignant cells. The defined mechanism of action for proteasome inhibitors has not been completely characterized, not all patients respond to proteasome inhibitor-based therapy, and inevitably patients develop resistance to proteasome inhibitors. Further investigation of the Ub+proteasome system (UPS) is needed to develop more effective inhibitors, to develop agents that overcome bortezomib resistance and to avoid adverse effects such as neuropathy. Furthermore, there are newly uncovered pathways, e.g., the SUMOylation and NEDDylation pathways, which similarly attach Ub-like proteins (ULPs) to protein substrates. The functional consequence of these modifications is only beginning to emerge, but these pathways have been linked to tumorigenesis and may similarly provide therapeutic targets. The immunoproteasome is a specialized form of the proteasome that produces peptides that are presented at the cell surface as major histocompatibility complex (MHC) class I antigens. Proteasome inhibitors decrease the presentation of antigenic peptides to reduce tumor cell recognition by cytotoxic T cells (CTLs) but unexpectedly increase tumor cell recognition by natural killer (NK) cells. Inhibitors of the UPS are validated, cytotoxic agents that may be further exploited in immunotherapy since they modulate tumor cell recognition by effectors of the immune system. Targeting the UPS, SUMOylation and NEDDylation pathways offers great promise in the treatment of hematologic and solid malignancies.

Strategies for the identification of novel inhibitors of deubiquitinating enzymes

Dysregulation of the UPS (ubiquitin-proteasome system) has been implicated in a wide range of pathologies including cancer, neurodegeneration and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; yet toxicity with this target remains high. DUBs (deubiquitinating enzymes) represent an alternative target in the UPS with low predicted toxicity. Currently, there are no DUB inhibitors that have been used clinically. To address this situation, Progenra has developed a novel assay to measure the proteolytic cleavage of Ub (ubiquitin) or UBL (Ub-like protein) conjugates such as SUMO (small Ub-related modifier), NEDD8 (neural-precursor-cell-expressed, developmentally down-regulated 8) or ISG15 (interferon-stimulated gene 15) by isopeptidases. In this review, current platforms for detecting DUB inhibitors are discussed and the advantages and disadvantages of the approaches are underlined.

ISG15 Inhibits Nedd4 Ubiquitin E3 Activity and Enhances the Innate Antiviral Response*♦

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral responses. The interferon-inducible ubiquitin-like protein ISG15 is expressed in cells in response to a variety of stress conditions like viral or bacterial infection and is present in its free form or is conjugated to cellular proteins. In addition, protein ubiquitination plays a regulatory role in the immune system. Many viruses modulate the ubiquitin (Ub) pathway to alter cellular signaling and the antiviral response. Ubiquitination of retroviral group-specific antigen precursors and matrix proteins of the Ebola, vesicular stomatitis, and rabies viruses by Nedd4 family HECT domain E3 ligases is an important step in facilitating viral release. We found that Nedd4 is negatively regulated by ISG15. Free ISG15 specifically bound to Nedd4 and blocked its interaction with Ub-E2 molecules, thus preventing further Ub transfer from E2 to E3. Furthermore, overexpression of ISG15 diminished the ability of Nedd4 to ubiquitinate viral matrix proteins and led to a decrease in the release of Ebola VP40 virus-like particles from the cells. These results point to a mechanistically novel function of ISG15 in the enhancement of the innate anti-viral response through specific inhibition of Nedd4 Ub-E3 activity. To our knowledge, this is the first example of a Ub-like protein with the ability to interfere with Ub-E2 and E3 interaction to inhibit protein ubiquitination.

The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis

In concert with the ubiquitin (Ub) proteasome system (UPS) the COP9 signalosome (CSN) controls the stability of cellular regulators. The CSN interacts with cullin-RING Ub ligases (CRLs) consisting of a specific cullin, a RING protein as Rbx1 and substrate recognition proteins. The Ub-like protein Nedd8 is covalently linked to cullins and removed by the CSN-mediated deneddylation. Cycles of neddylation and deneddylation regulate CRLs. Apoptotic stimuli cause caspase-dependent modifications of the UPS. However, little is known about the CSN during apoptosis. We demonstrate in vitro and in vivo that CSN6 is cleaved most effectively by caspase 3 at D23 after 2-3 h of apoptosis induced by anti-Fas-Ab or etoposide. CSN6 processing occurs in CSN-CRL complexes and is followed by the cleavage of Rbx1, the direct interaction partner of CSN6. Caspase-dependent cutting of Rbx1 is accompanied by decrease of neddylated proteins in Jurkat T cells. Another functional consequence of CSN6 cleavage is the enhancement of CSN-mediated deneddylating activity causing deneddylation of cullin 1 in cells. The CSN-associated deubiquitinating as well as kinase activity remained unchanged in presence of active caspase 3. The cleavage of Rbx1 and increased deneddylation of cullins inactivate CRLs and presumably stabilize pro-apoptotic factors for final apoptotic steps.