The immunoproteasome, the 20S proteasome and the PA28?? proteasome regulator are oxidative-stress-adaptive proteolytic complexes.
ABSTRACT: Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. We demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by 'pre-treatment' with very low concentrations of H2O2, and tested by measuring inducible resistance to a subsequent much higher 'challenge' dose of H2O2. Following an initial direct physical activation of pre-existing proteasomes, the 20S proteasome, immunoproteasome and PA28?? regulator all exhibited substantially increased de novo synthesis during adaptation over 24 h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and PA28?? synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and PA28 siRNA (short interfering RNA) knockdown treatments. Additionally, PA28??-knockout mutants achieved only half of the H2O2-induced adaptive increase in proteolytic capacity of wild-type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased, and oxidized proteins accumulated, during high H2O2 challenge, but prior H2O2 adaptation was protective. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or PA28?? regulator blocked 50-100% of these adaptive increases in cell division and DNA replication, and immunoproteasome knockdown largely abolished protection against protein oxidation.
Project description:The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (?1-?2-?5), the immunoproteasome (?1i-?2i-?5i) and the two intermediate proteasomes (?1-?2-?5i and ?1i-?2-?5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c--myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three ?5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, ?5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three ?5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.
Project description:In embryonic stem cells, removal of oxidatively damaged proteins is triggered upon the first signs of cell fate specification but the underlying mechanism is not known. Here, we report that this phase of differentiation encompasses an unexpected induction of genes encoding the proteasome activator PA28?? (11S), subunits of the immunoproteasome (20Si), and the 20Si regulator TNF?. This induction is accompanied by assembly of mature PA28-20S(i) proteasomes and elevated proteasome activity. Inhibiting accumulation of PA28? using miRNA counteracted the removal of damaged proteins demonstrating that PA28?? has a hitherto unidentified role required for resetting the levels of protein damage at the transition from self-renewal to cell differentiation.
Project description:The nuclear proteasome activator REGgamma/PA28gamma is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGgamma-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.
Project description:Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.
Project description:PA28? is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28? promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28?, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28?. Its recruitment to proteasome-containing complexes depends on PA28? and its expression increases the association of PA28? with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28?-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28? in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28? interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28? in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.
Project description:The proteasome activator PA28?? affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28??-iCP structure, how PA28?? regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28??-iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28?? and iCP through XL-MS. Our structures reveal a slight leaning of PA28?? towards the ?3-?4 side of iCP, disturbing the allosteric network of the gatekeeper ?2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28?? is distinct from those of constitutive CP by the homoheptameric TbPA26 or PfPA28. Our study sheds lights on the mechanism of enzymatic activity stimulation of immunoproteasome and suggests that PA28??-iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response.
Project description:Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C (Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H2O2) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female D. melanogaster (fruit-flies). Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H2O2, and blocking adaptive increases in proteasome expression also prevented full adaptation. We find, however, that this adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H2O2 adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keep1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.
Project description:All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles.
Project description:PA28 (also known as 11S, REG or PSME) is a family of proteasome regulators whose members are widely present in many of the eukaryotic supergroups. In jawed vertebrates they are represented by three paralogs, PA28?, PA28?, and PA28?, which assemble as heptameric hetero (PA28??) or homo (PA28?) rings on one or both extremities of the 20S proteasome cylindrical structure. While they share high sequence and structural similarities, the three isoforms significantly differ in terms of their biochemical and biological properties. In fact, PA28? and PA28? seem to have appeared more recently and to have evolved very rapidly to perform new functions that are specifically aimed at optimizing the process of MHC class I antigen presentation. In line with this, PA28?? favors release of peptide products by proteasomes and is particularly suited to support adaptive immune responses without, however, affecting hydrolysis rates of protein substrates. On the contrary, PA28? seems to be a slow-evolving gene that is most similar to the common ancestor of the PA28 activators family, and very likely retains its original functions. Notably, PA28? has a prevalent nuclear localization and is involved in the regulation of several essential cellular processes including cell growth and proliferation, apoptosis, chromatin structure and organization, and response to DNA damage. In striking contrast with the activity of PA28??, most of these diverse biological functions of PA28? seem to depend on its ability to markedly enhance degradation rates of regulatory protein by 20S proteasome. The present review will focus on the molecular mechanisms and biochemical properties of PA28?, which are likely to account for its various and complex biological functions and highlight the common features with the PA28?? paralog.
Project description:Intraneuronal accumulation of wild-type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full-length wild-type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse- and memory-associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP-response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2? (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2? with PA28? (a nuclear proteasome activator), the formation of PA28?-20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2? with 20S proteasome. Both downregulating PKAR2? by shRNA and upregulating proteasome by expressing PA28? rescued hTau-induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau-induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28?-20S proteasome complex formation contributes to PKAR2? elevation and PKA inhibition.