Nrf2-dependent induction of proteasome and Pa28αβ regulator are required for adaptation to oxidative stress

The ability to adapt to acute oxidative stress (e.g. H(2)O(2), peroxynitrite, menadione, and paraquat) through transient alterations in gene expression is an important component of cellular defense mechanisms. We show that such adaptation includes Nrf2-dependent increases in cellular capacity to degrade oxidized proteins that are attributable to increased expression of the 20 S proteasome and the Pa28αβ (11 S) proteasome regulator. Increased cellular levels of Nrf2, translocation of Nrf2 from the cytoplasm to the nucleus, and increased binding of Nrf2 to antioxidant response elements (AREs) or electrophile response elements (EpREs) in the 5'-untranslated region of the proteasome β5 subunit gene (demonstrated by chromatin immunoprecipitation (or ChIP) assay) are shown to be necessary requirements for increased proteasome/Pa28αβ levels, and for maximal increases in proteolytic capacity and stress resistance; Nrf2 siRNA and the Nrf2 inhibitor retinoic acid both block these adaptive changes and the Nrf2 inducers DL-sulforaphane, lipoic acid, and curcumin all replicate them without oxidant exposure. The immunoproteasome is also induced during oxidative stress adaptation, contributing to overall capacity to degrade oxidized proteins and stress resistance. Two of the three immunoproteasome subunit genes, however, contain no ARE/EpRE elements, and Nrf2 inducers, inhibitors, and siRNA all have minimal effects on immunoproteasome expression during adaptation to oxidative stress. Thus, immunoproteasome appears to be (at most) minimally regulated by the Nrf2 signal transduction pathway.     

Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC–MS

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20–30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.     

Differential roles of proteasome and immunoproteasome regulators Pa28αβ, Pa28γ and Pa200 in the degradation of oxidized proteins

The response and functions of proteasome regulators Pa28αβ (or 11S), Pa28γ and Pa200 in oxidative-stress adaptation (also called hormesis) was studied in murine embryonic fibroblasts (MEFs), using a well-characterized model of cellular adaptation to low concentrations (1.0-10.0 μM) of hydrogen peroxide (H(2)O(2)), which alter gene expression profiles, increasing resistance to higher levels of oxidative-stress. Pa28αβ bound to 20S proteasomes immediately upon H(2)O(2)-treatment, whereas 26S proteasomes were disassembled at the same time. Over the next 24h, the levels of Pa28αβ, Pa28γ and Pa200 proteasome regulators increased during H(2)O(2)-adaptation, whereas the 19S regulator was unchanged. Purified Pa28αβ, and to a lesser extent Pa28γ, significantly increased the ability of purified 20S proteasome to selectively degrade oxidized proteins; Pa28αβ also increased the capacity of purified immunoproteasome to selectively degrade oxidized proteins but Pa28γ did not. Pa200 regulator actually decreased 20S proteasome and immunoproteasome's ability to degrade oxidized proteins but Pa200 and poly-ADP ribose polymerase may cooperate in enabling initiation of DNA repair. Our results indicate that cytoplasmic Pa28αβ and nuclear Pa28γ may both be important regulators of proteasome's ability to degrade oxidatively-damaged proteins, and induced-expression of both 20S proteasome and immunoproteasome, and their Pa28αβ and Pa28γ regulators are important for oxidative-stress adaptation.     

The proteasome system in infection: impact of β5 and LMP7 on composition, maturation and quantity of active proteasome complexes

Proteasomes are the major enzyme complexes for non-lysosomal protein degradation in eukaryotic cells. Mammals express two sets of catalytic subunits: the constitutive subunits β1, β2 and β5 and the immunosubunits LMP2 (β1i), MECL-1 (β2i) and LMP7 (β5i). The LMP7-propeptide (proLMP7) is required for optimal maturation of LMP2/MECL-1-containing precursors to mature immunoproteasomes, but can also mediate efficient integration into mixed proteasomes containing β1 and β2. In contrast, the β5-propeptide (proβ5) has been suggested to promote preferential integration into β1/β2-containing precursors, consequently favouring the formation of constitutive proteasomes. Here, we show that proβ5 predominantly promotes integration into LMP2/MECL-1-containing precursors in IFNγ-stimulated, LMP7-deficient cells and infected LMP7-deficient mice. This demonstrates that proβ5 does not direct preferential integration into β1/β2-containing precursors, but instead promotes the formation of mixed LMP2/MECL-1/β5 proteasomes under inflammatory conditions. Moreover, the propeptides substantially differ in their capacity to promote proteasome maturation, with proLMP7 showing a significantly higher chaperone activity as compared to proβ5. Increased efficiency of proteasome maturation mediated by proLMP7 is required for optimal MHC class I cell surface expression and is equally important as the catalytic activity of immunoproteasomes. Intriguingly, induction of LMP7 by infection not only results in rapid exchange of constitutive by immunosubunits, as previously suggested, but also increases the total proteasome abundance within the infected tissue. Hence our data identify a novel LMP7-dependend mechanism to enhance the activity of the proteasome system in infection, which is based on the high chaperone activity of proLMP7 and relies on accelerated maturation of active proteasome complexes.     

Structure and functional analyses of the 26S proteasome subunits from plants – Plant 26S proteasome

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10 strains which grow normally, Physcomitrella rpn10 strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.     

Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs), a highly cytotoxic DNA lesion, activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.Key words: genomic stability, DNA repair, double-strand breaks, ATM, proteasome, PA28γ (PSME3)     

Phenoxypropanolamine derivatives as selective inhibitors of the 20S proteasome β1 and β5 subunits

New series of thiophene-containing phenoxypropanolamines were synthesized and evaluated for their potency to inhibit the three proteolytic activities of the mammalian 20S proteasome. Noticeable inhibition of both ChT-L and PA activities was obtained with three compounds: one with unsubstituted phenoxypropanolamine group (7) and the two others with a p-Cl-substituted group (4 and 9). For three other compounds (3, 8 and 10), ChT-L activity alone was significantly inhibited. In silico docking performed on the β5 and β1 subunits bearing the respective ChT-L and PA catalytic sites showed features common to poses associated with active compounds. These features may constitute a selectivity criterion for structure-guided inhibitor design.     

Accelerated formation of α-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations

A hallmark of Parkinson disease (PD) is the formation of intracellular protein inclusions called Lewy bodies that also contain mitochondria. α-Synuclein (αSyn) is a major protein component of Lewy bodies, where it is in an amyloid conformation and a significant fraction is truncated by poorly understood proteolytic events. Previously, we demonstrated that the 20S proteasome cleaves αSyn in vitro to produce fragments like those observed in Lewy bodies and that the fragments accelerate the formation of amyloid fibrils from full-length αSyn. Three point mutations in αSyn are associated with early-onset familial PD: A30P, E46K, and A53T. However, these mutations have very different effects on the amyloidogenicity and vesicle-binding activity of αSyn, suggesting neither of these processes directly correlate with neurodegeneration. Here, we evaluate the effect of the disease-associated mutations on the fragmentation, conformation, and association reactions of αSyn in the presence of the 20S proteasome and liposomes. The 20S proteasome produced the C-terminal fragments from both the mutant and wildtype αSyn. These truncations accelerated fibrillization of all α-synucleins, but again there was no clear correlation between the PD-associated mutations and amyloid formation in the presence of liposomes. Recent data suggests that cellular toxicity is caused by a soluble oligomeric species, which is a precursor to the amyloid form and is immunologically distinguishable from both soluble monomeric and amyloid forms of αSyn. Notably, the rate of formation of the soluble, presumptively cytotoxic oligomers correlated with the disease-associated mutations when both 20S proteasome and liposomes were present. Under these conditions, the wildtype protein was also cleaved and formed the oligomeric structures, albeit at a slower rate, suggesting that 20S-mediated truncation of αSyn may play a role in sporadic PD as well. Evaluation of the biochemical reactions of the PD-associated α-synuclein mutants in our in vitro system provides insight into the possible pathogenetic mechanism of both familial and sporadic PD.     

The COP9 signalosome: an alternative lid for the 26S proteasome?

Two groups have recently reported ubiquitin-deconjugating activities associated with the COP9 signalosome (CSN). Together with a previously identified deneddylation activity, CSN now appears to possess biochemical activities towards the cullin-containing ubiquitin ligases (e.g. deneddylation) and ubiquitinated protein substrates in the proteasome-mediated degradation processes (e.g. deubiquitination). Here, we discuss how these findings support a central role for CSN in ubiquitination of substrate proteins and their proteolysis     

To misfold or to lose structure?: Detection and degradation of oxidized proteins by the 20S proteasome

Aggregation of proteins damaged by stress is often a causal factor of cell death. To prevent aggregation, eukaryotic cells rapidly degrade damaged proteins by engaging two types of proteasomes. The first type is the 26S proteasome (26SP) which is composed of a cylindrical proteolytic core—the 20S proteasome (20SP)—and one or two regulatory particles (RPs) that interact with ubiquitinated proteins. The second type is the free 20SP which mediates ubiquitin-independent proteolysis. We have recently shown that loss of RP function in Arabidopsis leads to an expected decrease in 26SP-dependent protein degradation and hypersensitivity to stresses that induce protein misfolding. Surprisingly, RP mutants have increased 20SP activity and tolerance to oxidative stress. This finding suggests that misfolded proteins carry one type of degradation signal that steers them to ubiquitination enzymes and the 26SP, while oxidatively damaged proteins carry another that guides them directly to the 20SP for degradation. Here we suggest that protein oxidation induces the formation of unstructured regions that serve as targeting signals for 20SP-dependent proteolysis.Key words: 20S proteasome, misfolded proteins, oxidized proteins, ubiquitin-independent proteolysis, unstructured regionsProteasomes are an essential component of the quality-control system that limits the accumulation of non-functional proteins in the cell.^1–4 A protein can be rendered non-functional by mutations, translational and folding errors, and adverse conditions such as heat shock and oxidative stress. These proteins decrease the efficiency of metabolic pathways not only because of their loss of function, but also because of the deleterious gain-of-function effects generally known as proteotoxicity.^1–4 Until recently, it was widely accepted that the detection and degradation of all non-functional proteins is initiated by their loss of native tertiary structure followed by misfolding. Misfolding is believed to expose hydrophobic regions that form interaction domains for chaperones, which are in turn bound to ubiquitin ligases that label the target for 26SP-dependent proteolysis.^5–9 Thus, the degradation of proteins that have lost their native conformation was considered to be an ubiquitin (Ub)- and 26SP-dependent process (Fig. 1).Open in a separate windowFigure 1Model for the degradation of damaged proteins. (A) Stresses such as heat shock or the incorporation of amino acid analogues induce protein misfolding. If for example, a globular protein is misfolded, its hydrophobic core will be exposed to the cytoplasm. These hydrophobic regions can bind chaperones that either repair the misfolded protein or shuttle it to the ubiquitination enzymes and the 26SP. (B) Protein oxidation leads to a partial loss of secondary structure without disrupting the overall folding pattern of the protein, resulting in flexible, unstructured regions. These regions serve as degradation signals for the Ub-independent 20SP pathway.However, a number of studies have shown that the degradation of proteins damaged by oxidative stress follows another route: oxidized proteins are degraded by the 20SP in a Ub-independent manner.^5–7,10 We have recently shown that this proteolysis pathway is important for oxidative stress tolerance in plants. Loss of function of the Arabidopsis RP subunits RPT2a, RPN10 and RPN12a reduces 26SP function and leads to an expected decrease in Ub-dependent proteolysis.^11–14 Unexpectedly, all three RP mutants have increased 20SP activity, which is probably caused by the stabilization of an activator of proteasome biogenesis that is normally degraded by the 26SP.¹¹ This shift in proteasome activity leads to increased oxidized protein turnover and oxidative stress tolerance, but also to decreased tolerance to stresses that are known to cause protein misfolding.¹¹ Thus, the 26SP in Arabidopsis is needed for the removal of misfolded proteins, and the 20SP is essential for the degradation of oxidized proteins.This differential degradation of damaged proteins implies that plant cells have distinct recognition mechanisms for misfolded and oxidized proteins, and that oxidation leads to the formation of a specific degradation signal that channels the oxidized proteins directly to the 20SP. Nevertheless, it has been suggested that the proteolysis of oxidized proteins also depends on misfolding and exposure of hydrophobic regions that serve as recognition sites for either the 20SP itself or for specific chaperonins that bind the 20SP.^5–7,10,15 If the recognition of proteasomal targets is specific, then—according to the current theory—heat shock and oxidative stress would expose specific types of hydrophobic degradation signals in any cellular protein. These qualitatively different hydrophobic regions would lead either to ubiquitination and 26SP-dependent degradation or to a direct interaction with the 20SP. While we cannot exclude this, it is hard to envision how a random process such as misfolding would produce discernable degradation signals dependent on whether the denaturation was caused by heat shock or by oxidation. An alternative explanation is that the recognition of oxidized proteins does not depend on misfolding.How would oxidized proteins then be targeted to the 20SP? Today we know of some functional proteins that are degraded by the 20SP in a Ub-independent manner, and all these characterized 20SP targets have regions that lack secondary structure.^10,16 The native unstructured regions or intrinsically disordered regions give conformational plasticity to a protein and allow it to form a complex with different partners.^17,18 The unstructured regions are also thought to serve as initiation sites for proteolysis.¹⁹ These findings are a starting point for the “degradation by default” theory which states that many proteins in their native conformation contain unstructured regions that make them inherently unstable and target them to the Ub-independent 20SP pathway.¹⁰ Such proteins tend to be stabilized by forming complexes in which the unstructured regions are masked by other polypeptides. Since oxidized proteins are processed by the 20SP, their common degradation signal could also be an intrinsically disordered region (Fig. 1). Thus, protein oxidation—at least mild protein oxidation⁶—would lead to the formation of flexible peptide stretches (i.e., unstructured regions) rather than to protein misfolding (i.e., unfolding and non-native refolding that exposes otherwise sequestered hydrophobic residues). This hypothesis is supported by a study of the 20SP-dependent degradation of oxidized calmodulin (CaM).²⁰ Oxidation of CaM leads to a significant increase in its 20SP-dependent and Ub-independent degradation. In vitro studies revealed a positive correlation between decreased secondary structure (i.e., increased flexibility) and proteolysis rate, and no correlation between changes in surface hydrophobicity and CaM stability.²⁰There is another paradox concerning 20SP-dependent proteolysis of oxidized proteins. The 20SP is a barrel-shaped particle composed of two α and two β rings in an α7β7b7β7 configuration.²¹ Proteolytic activity is confined to the β rings and is broad range, so that it degrades any target into oligopeptides of 3–25 amino acids in length. To be degraded, targets must not only be recognized by the 20SP, but must also enter into the proteolytic chamber through a constriction in the α rings known as the α-annulus. In 26SP-dependent proteolysis, this entry point is opened by the action of a ring of AAA ATPases from the RP.²¹ However, this entrance gate of the free 20SP is closed and restricts random proteolysis. How then do oxidized proteins enter the proteolytic chamber? It has been shown that some natively unstructured proteins can open the gates possibly by acting as chaotropes and by causing subunit residues to become disordered.²² This then could also be the entry mechanism for oxidized proteins.In conclusion, analyses of Arabidopsis proteasome mutants with decreased Ub-dependent proteolysis reveals that the 20SP-dependent “degradation by the default” pathway is operational in plants and is important for oxidative stress tolerance. However, it remains to be shown whether indeed the unstructured regions, either innate or formed by the action of free radicals, guide proteins to the 20SP and specifically cause the opening of the α-annulus. The identities of native 20SP targets in plants also await further studies.     

The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.     

Fusion of vacuoles--where are the membranes, and where are the holes?

In the February 8th issue of Cell, Wang et al. report the surprising finding that vacuolar fusion occurs at the periphery of the contact area of the vacuoles and not by the expansion of a central fusion pore. During fusion, a disk of boundary membrane is excised and left behind within the fused vacuoles.     

Proteolytic degradation of heme-modified hepatic cytochromes P450: A role for phosphorylation, ubiquitination, and the 26S proteasome?

The resident integral hepatic endoplasmic reticulum (ER) proteins, cytochromes P450 (P450s), turn over in vivo with widely varying half-lives. We and others (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992; and Tierney et al., Arch. Biochem. Biophys. 293, 9, 1992) have previously shown that in intact animals, the hepatic P450s of the 3A and 2E1 subfamilies are first ubiquitinated and then proteolyzed after their drug-induced suicide inactivation. Our findings with intact rat hepatocytes and ER preparations containing native P450s and P450s inactivated via heme modification of the protein have revealed that the proteolytic degradation of heme-modified P450s requires a cytosolic ATP-dependent proteolytic system rather than lysosomal or ER proteases (Correia et al., Arch. Biochem. Biophys. 297, 228, 1992). Using purified cumene hydroperoxide-inactivated P450s (rat liver P450s 2B1 or 3A and/or a recombinant human liver P450 3A4) as models, we now document that these heme-modified enzymes are indeed ubiquitinated and then proteolyzed by the 26S proteasome, but not by its 20S proteolytic core. In addition, our studies indicate that the ubiquitination of these heme-modified P450s is preceded by their phosphorylation. It remains to be determined whether, in common with several other cellular proteins, such P450 phosphorylation is indeed required for their degradation. Nevertheless, these findings suggest that the membrane-anchored P450s are to be included in the growing class of ER proteins that undergo ubiquitin-dependent 26S proteasomal degradation.