Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress.

Interferon (IFN)-induced immunoproteasomes (i-proteasomes) have been associated with improved processing of major histocompatibility complex (MHC) class I antigens. Here, we show that i-proteasomes function to protect cell viability under conditions of IFN-induced oxidative stress. IFNs trigger the production of reactive oxygen species, which induce protein oxidation and the formation of nascent, oxidant-damaged proteins. We find that the ubiquitylation machinery is concomitantly upregulated in response to IFNs, functioning to target defective ribosomal products (DRiPs) for degradation by i-proteasomes. i-proteasome-deficiency in cells and in murine inflammation models results in the formation of aggresome-like induced structures and increased sensitivity to apoptosis. Efficient clearance of these aggregates by the enhanced proteolytic activity of the i-proteasome is important for the preservation of cell viability upon IFN-induced oxidative stress. Our findings suggest that rather than having a specific role in the production of class I antigens, i-proteasomes increase the peptide supply for antigen presentation as part of a more general role in the maintenance of protein homeostasis.

Nedd8 processing enzymes in Schizosaccharomyces pombe.

BACKGROUND: Conjugation of the ubiquitin-like modifier Nedd8 to cullins is critical for the function of SCF-type ubiquitin ligases and thus facilitates ubiquitin conjugation and ultimately degradation of SCF substrates, including several cell cycle regulators. Like ubiquitin, Nedd8 is produced as a precursor that must first be processed before it becomes active. In Saccharomyces cerevisiae this is carried out exclusively by the enzyme Yuh1. RESULTS: Here we show that in the fission yeast, Schizosaccharomyces pombe, the Yuh1 orthologue, Uch1, is not the sole Nedd8 processing enzyme. Instead it appears that deubiquitylating enzymes can efficiently process the Nedd8 precursor in vivo. CONCLUSIONS: Several enzymes contribute to Nedd8 precursor processing including a number of deubiquitylating enzymes.

Ubiquitin system: JAMMing in the name of the lid.

The isopeptide bonds formed by ubiquitin or its relatives are cleaved by hydrolases with active site cysteines. Recent studies have revealed that similar metalloprotease motifs--JAMMs--in the Rpn11 subunit of the 26S proteasome lid and in the Csn5 subunit of the COP9 signalosome are involved in deubiquitination and deneddylation, respectively.

Consequences of COP9 signalosome and 26S proteasome interaction.

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr-Pad1-N-terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the formation of an intact Flag-CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull-downs also precipitated cullins indicating the existence of super-complexes consisting of the CSN, the 26S proteasome and cullin-based Ub ligases. Permanent expression of a chimerical subunit (Flag-CSN2-Rpn6) consisting of the N-terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag-CSN2 overexpression was de-novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c-Jun in B8 cells. The possible role of super-complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.

Purification method of the COP9 signalosome from human erythrocytes.

The COP9 signalosome (CSN) is a multimeric protein complex that occurs in all eukaryotic cells. Originally described in plants as a regulator of photomorphogenesis, its purification and characterization from mammalian cells revealed significant sequence homologies to subunits of the 26S proteasome lid complex, as well as of the eukaryotic translation initiation factor 3. Recent studies disclosed its participation in processes such as DNA repair, cell cycle regulation, development, and angiogenesis. At the moment, the pleiotropic effects of the CSN point to a regulatory role in the ubiquitin/26S proteasome system, but its exact function still remains to be clarified. This chapter describes the method to purify human CSN from red blood cells. Two outdated erythrocyte concentrates are sufficient to prepare approximately 0.5 mg of CSN. Washed cells are first lysed and then proteins are separated by a DEAE anion-exchange column. The CSN-containing fractions are pooled and subjected to an ammonium sulfate precipitation followed by dialysis. The concentrated proteins are then loaded onto a glycerol density gradient and ultracentrifugation is performed. The purification procedure is continued using two succeeding anion-exchange columns, resulting in a sufficiently pure CSN complex. Optionally, an additional density gradient centrifugation can be attached. The purified CSN complex possesses kinase, deneddylase, and deubiquitinase activities and can be stored for at least 2 months on ice at 4 degrees .

Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast.

Conjugation of proteins to ubiquitin plays a central role for a number of cellular processes including endocytosis, DNA repair and degradation by the 26S proteasome. However, ubiquitination is reversible as a number of deubiquitinating enzymes mediate the disassembly of ubiquitin-protein conjugates. Some deubiquitinating enzymes are associated with the 26S proteasome contributing to and regulating the particle's activity. Here, we characterise fission yeast Uch2 and Ubp6, two proteasome associated deubiquitinating enzymes. The human orthologues of these enzymes are known as Uch37 and Usp14, respectively. We report that the subunit Uch2/Uch37 is the major deubiquitinating enzyme associated with the fission yeast 26S proteasome. In contrast, the activity of Ubp6 appears to play a more regulatory and/or structural role involving the proteasome subunits Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12, as Ubp6 becomes essential when activity of these subunits is compromised by conditional mutations. Finally, when the genes encoding Uch2/Uch37 and Ubp6 are disrupted, the cells are viable without showing obvious signs of impaired ubiquitin-dependent proteolysis, indicating that other deubiquitinating enzymes may remedy for the redundancy of these enzymes.

Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome.

The 26S proteasome degrades polyubiquitylated (polyUb) proteins by an ATP-dependent mechanism. Here we show that binding of model polyUb substrates to the 19S regulator of mammalian and yeast 26S proteasomes enhances the peptidase activities of the 20S proteasome about two-fold in a process requiring ATP hydrolysis. Monoubiquitylated proteins or tetraubiquitin alone exert no effect. However, 26S proteasomes from the yeast alpha3DeltaN open-gate mutant and the rpt2YA and rpt5YA mutants with impaired gating can still be activated (approximately 1.3-fold to 1.8-fold) by polyUb-protein binding. Thus, binding of polyUb substrates to the 19S regulator stabilizes gate opening of the 20S proteasome and induces conformational changes of the 20S proteasome that facilitate channeling of substrates and their access to active sites. In consequence, polyUb substrates will allosterically stimulate their own degradation.

Intracellular IL-1 receptor antagonist type 1 inhibits IL-1-induced cytokine production in keratinocytes through binding to the third component of the COP9 signalosome.

The IL-1 receptor antagonist (IL-1Ra) exists in four isoforms, three of which lack signal peptides and are primarily intracellular proteins. The biologic roles of the intracellular isoforms of IL-1Ra have remained unknown. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra 18-kDa type 1 (icIL-1Ra1), mediated unique functions inside cells. A yeast two-hybrid screen with HeLa cell lysates revealed specific binding of icIL-1Ra1, and not of the other IL-1Ra isoforms, to the third component of the COP9 signalosome complex (CSN3). This binding was confirmed by Far Western blot analysis, sedimentation on a glycerol gradient, glutathione pull-down experiments, and coimmunoprecipitation. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of p53, c-Jun, and IkappaB by the crude CSN-associated kinase and of p53 by recombinant protein kinase CK2 and protein kinase D, both associated with CSN3. The biologic relevance of the interaction between icIL-1Ra1 and CSN3 was demonstrated in the keratinocyte cell lines KB and A431, both possessing abundant CSN3. A431 cells exhibited high levels of icIL-1Ra1 but lacked both detectable IL-1alpha-induced IL-6 and IL-8 production and phosphorylation of p38 MAPK. KB cells displayed the opposite pattern which was reversed after transfection with icIL-1Ra1 mRNA. Inhibition of CSN3 or of icIL-1Ra1 production through gene knockdown with specific small interfering RNA in A431 cells each led to an inhibition of IL-1alpha-induced IL-6 and IL-8 production. Thus, icIL-1Ra1 exhibits unique anti-inflammatory properties inside cells through binding to CSN3 with subsequent inhibition of the p38 MAPK signal transduction pathway.

The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis.

Recently the COP9 signalosome (CSN) has become a focus of interest for many researchers, because of its function at the interface between signal transduction and ubiquitin-dependent proteolysis. It is required for the proper progression of the cell cycle in Schizosaccharomyces pombe and is essential for development in plants and Drosophila. However, its function in mammalian cells remains obscure. Although the CSN shares structural similarities with the 26S proteasome lid complex (LID), its functions seem to be different from that of the LID. A variety of CSN-specific protein-protein interactions have been described in mammalian cells. However, it is currently unclear how many reflect true functions of the complex. Two activities associated with the CSN have been identified so far: a protein kinase and a deneddylase. The CSN-associated kinase phosphorylates transcription factors, which determines their stability towards the ubiquitin system. The associated deneddylase regulates the activity of specific SCF E3 ubiquitin ligases. The CSN thus appears to be a platform connecting signalling with proteolysis.

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome.

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to DeltaCSN3(111-403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun-Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.