PA28γ, the ring that makes tumors invisible to the immune system?

PA28γ is a proteasomal interactor whose main and most known function is to stimulate the hydrolytic activity of the 20 S proteasome independently of ubiquitin and ATP. Unlike its two paralogues, PA28α and PA28ß, PA28γ is largely present in the nuclear compartment and plays pivotal functions in important pathways such as cellular division, apoptosis, neoplastic transformation, chromatin structure and organization, fertility, lipid metabolism, and DNA repair mechanisms. Although it is known that a substantial fraction of PA28γ is found in the cell in a free form (i.e. not associated with 20 S), almost all of the studies so far have focused on its ability to modulate proteasomal enzymatic activities. In this respect, the ability of PA28γ to strongly stimulate degradation of proteins, especially if intrinsically disordered and therefore devoid of three-dimensional tightly folded structure, appears to be the main molecular mechanism underlying its multiple biological effects. Initial studies, conducted more than 20 years ago, came to the conclusion that among the many biological functions of PA28γ, the immunological ones were rather limited and circumscribed. In this review, we focus on recent evidence showing that PA28γ fulfills significant functions in cell-mediated acquired immunity, with a particular role in attenuating MHC class I antigen presentation, especially in relation to neoplastic transformation and autoimmune diseases.

Targeting immunoproteasome in neurodegeneration: A glance to the future.

The immunoproteasome is a specialized form of proteasome equipped with modified catalytic subunits that was initially discovered to play a pivotal role in MHC class I antigen processing and immune system modulation. However, over the last years, this proteolytic complex has been uncovered to serve additional functions unrelated to antigen presentation. Accordingly, it has been proposed that immunoproteasome synergizes with canonical proteasome in different cell types of the nervous system, regulating neurotransmission, metabolic pathways and adaptation of the cells to redox or inflammatory insults. Hence, studying the alterations of immunoproteasome expression and activity is gaining research interest to define the dynamics of neuroinflammation as well as the early and late molecular events that are likely involved in the pathogenesis of a variety of neurological disorders. Furthermore, these novel functions foster the perspective of immunoproteasome as a potential therapeutic target for neurodegeneration. In this review, we provide a brain and retina-wide overview, trying to correlate present knowledge on structure-function relationships of immunoproteasome with the variety of observed neuro-modulatory functions.

A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose.

BACKGROUND: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. RESULTS: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1ß and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit. CONCLUSIONS: This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

Induction by Phenobarbital of Phase I and II Xenobiotic-Metabolizing Enzymes in Bovine Liver: An Overall Catalytic and Immunochemical Characterization.

In cattle, phenobarbital (PB) upregulates target drug-metabolizing enzyme (DME) mRNA levels. However, few data about PB's post-transcriptional effects are actually available. This work provides the first, and an almost complete, characterization of PB-dependent changes in DME catalytic activities in bovine liver using common probe substrates and confirmatory immunoblotting investigations. As expected, PB increased the total cytochrome P450 (CYP) content and the extent of metyrapone binding; moreover, an augmentation of protein amounts and related enzyme activities was observed for known PB targets such as CYP2B, 2C, and 3A, but also CYP2E1. However, contradictory results were obtained for CYP1A, while a decreased catalytic activity was observed for flavin-containing monooxygenases 1 and 3. The barbiturate had no effect on the chosen hydrolytic and conjugative DMEs. For the first time, we also measured the 26S proteasome activity, and the increase observed in PB-treated cattle would suggest this post-translational event might contribute to cattle DME regulation. Overall, this study increased the knowledge of cattle hepatic drug metabolism, and further confirmed the presence of species differences in DME expression and activity between cattle, humans, and rodents. This reinforced the need for an extensive characterization and understanding of comparative molecular mechanisms involved in expression, regulation, and function of DMEs.

Regulating Proteasome Activity.

Strictly controlled degradation of the proteome is a key factor in maintaining cellular homeostasis and allows a rapid and effective response to a variety of different stress challenges [...].

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the ß5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

PA28γ-20S proteasome is a proteolytic complex committed to degrade unfolded proteins.

PA28γ is a nuclear activator of the 20S proteasome that, unlike the 19S regulatory particle, stimulates hydrolysis of several substrates in an ATP- and ubiquitin-independent manner and whose exact biological functions and molecular mechanism of action still remain elusive. In an effort to shed light on these important issues, we investigated the stimulatory effect of PA28γ on the hydrolysis of different fluorogenic peptides and folded or denatured full-length proteins by the 20S proteasome. Importantly, PA28γ was found to dramatically enhance breakdown rates by 20S proteasomes of several naturally or artificially unstructured proteins, but not of their native, folded counterparts. Furthermore, these data were corroborated by experiments in cell lines with a nucleus-tagged myelin basic protein. Finally, mass spectrometry analysis of the products generated during proteasomal degradation of two proteins demonstrated that PA28γ does not increase, but rather decreases, the variability of peptides that are potentially suitable for MHC class I antigen presentation. These unexpected findings indicate that global stimulation of the degradation of unfolded proteins may represent a more general feature of PA28γ and suggests that this proteasomal activator might play a broader role in the pathway of protein degradation than previously believed.

PA28γ: New Insights on an Ancient Proteasome Activator.

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, PA28α, PA28ß, and PA28γ, which assemble as heptameric hetero (PA28αß) or homo (PA28γ) 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, PA28α and PA28ß 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, PA28αß 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, PA28γ 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, PA28γ 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 PA28αß, most of these diverse biological functions of PA28γ 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 PA28γ, which are likely to account for its various and complex biological functions and highlight the common features with the PA28αß paralog.

Tumors escape immunosurveillance by overexpressing the proteasome activator PSME3.

The success of CD8+ T cell-based cancer immunotherapy emphasizes the importance of understanding the mechanisms of generation of MHC-I peptide ligands and the possible pathways of tumor cell escape from immunosurveillance. Recently, we showed that peptides generated in the nucleus during a pioneer round of mRNA translation (pioneer translation products, or PTPs) are an important source of tumor specific peptides which correlates with the aberrant splicing and transcription events associated with oncogenesis. Here we show that up-regulation of PSME3 proteasome activator in cancer cells results in increased destruction of PTP-derived peptides in the nucleus thus enabling cancer cell to subvert immunosurveillance. These findings unveil a previously unexpected role for PSME3 in antigen processing and identify PSME3 as a druggable target to improve the efficacy of cancer immunotherapy.

Iron Causes Lipid Oxidation and Inhibits Proteasome Function in Multiple Myeloma Cells: A Proof of Concept for Novel Combination Therapies.

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

Proteasome stress sensitizes malignant pleural mesothelioma cells to bortezomib-induced apoptosis.

Based on promising results in preclinical models, clinical trials have been performed to evaluate the efficacy of the first-in-class proteasome inhibitor bortezomib towards malignant pleural mesothelioma (MPM), an aggressive cancer arising from the mesothelium of the serous cavities following exposure to asbestos. Unexpectedly, only minimal therapeutic benefits were observed, thus implicating that MPM harbors inherent resistance mechanisms. Identifying the molecular bases of this primary resistance is crucial to develop novel pharmacologic strategies aimed at increasing the vulnerability of MPM to bortezomib. Therefore, we assessed a panel of four human MPM lines with different sensitivity to bortezomib, for functional proteasome activity and levels of free and polymerized ubiquitin. We found that highly sensitive MPM lines display lower proteasome activity than more bortezomib-resistant clones, suggesting that reduced proteasomal capacity might contribute to the intrinsic susceptibility of mesothelioma cells to proteasome inhibitors-induced apoptosis. Moreover, MPM equipped with fewer active proteasomes accumulated polyubiquitinated proteins, at the expense of free ubiquitin, a condition known as proteasome stress, which lowers the cellular apoptotic threshold and sensitizes mesothelioma cells to bortezomib-induced toxicity as shown herein. Taken together, our data suggest that an unfavorable load-versus-capacity balance represents a critical determinant of primary apoptotic sensitivity to bortezomib in MPM.

The amyloidogenic light chain is a stressor that sensitizes plasma cells to proteasome inhibitor toxicity.

Systemic light chain (AL) amyloidosis is caused by the clonal production of an unstable immunoglobulin light chain (LC), which affects organ function systemically. Although pathogenic LCs have been characterized biochemically, little is known about the biology of amyloidogenic plasma cells (PCs). Intrigued by the unique response rates of AL amyloidosis patients to the first-in-class proteasome inhibitor (PI) bortezomib, we purified and investigated patient-derived AL PCs, in comparison with primary multiple myeloma (MM) PCs, the prototypical PI-responsive cells. Functional, biochemical, and morphological characterization revealed an unprecedented intrinsic sensitivity of AL PCs to PIs, even higher than that of MM PCs, associated with distinctive organellar features and expression patterns indicative of cellular stress. These consisted of expanded endoplasmic reticulum (ER), perinuclear mitochondria, and a higher abundance of stress-related transcripts, and were consistent with reduced autophagic control of organelle homeostasis. To test whether PI sensitivity stems from AL LC production, we engineered PC lines that can be induced to express amyloidogenic and nonamyloidogenic LCs, and found that AL LC expression alters cell growth and proteostasis and confers PI sensitivity. Our study discloses amyloidogenic LC production as an intrinsic PC stressor, and identifies stress-responsive pathways as novel potential therapeutic targets. Moreover, we contribute a cellular disease model to dissect the biology of AL PCs.

Low proviral small ruminant lentivirus load as biomarker of natural restriction in goats.

Small ruminant lentiviruses (SRLV) globally affect welfare and production of sheep and goats and are mainly controlled through elimination of infected animals, independently of the viral kinetics within the single animal. Control programs are based on highly sensitive serological tests, however the existence of low antibody responders leads to the permanent presence of seronegative infected animals in the flock, thus perpetuating the infection. On the other hand, long-term non-progressors show a detectable antibody response not indicative of a shedding animal, suggesting immune contention of infection. In this study, we analyse two goat populations within the same herd, harbouring low or high proviral SRLV loads respectively, both showing a robust antibody response. In vivo findings were confirmed in vitro since fibroblastic cell lines obtained from one high and one low proviral load representative goats, showed respectively a high and a faint production of virus upon infection with reference and field circulating SRLV strains. Differences in virus production were relieved when strain CAEV-Co was used for experimental infection. We analysed LTR promoter activity, proviral load, entry step and production of virus and viral proteins. Intriguingly, proteasomal activity was higher in fibroblasts from low proviral load animals and proteasome inhibition increased viral production in both cell lines, suggesting the implication of active proteasome-dependent restriction factors. Among them, we analysed relative expression and sequences of TRIM5α, APOBEC3 (Z1, Z2, Z3 and Z2-Z3) and BST-2 (Tetherin) and found a global antiviral status in low proviral carriers that may confer protection against viral shedding and disease onset.

A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells.

Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.

Comparative study of the biochemical properties of proteasomes in domestic animals.

Information on the biochemical properties of proteasomes is lacking or, at best, only fragmentary for most species of veterinary interest. Moreover, direct comparison of the limited data available on the enzymatic features of proteasomes in domestic animals is rendered difficult due to the heterogeneity of the experimental settings used. This represents a clear drawback in veterinary research, given the crucial involvement of proteasomes in control of several physiological and pathological processes. We performed the first comparative analysis of key biochemical properties of proteasomes obtained from 8 different domestic mammals. Specifically, we investigated the three main peptidase activities of constitutive and immunoproteasomes in parallel and systematically checked the sensitivity of the chymotryptic site to three of the most potent and selective inhibitors available. Overall, there was substantial similarity in the enzymatic features of proteasomes among the species examined, although some interesting species-specific features were observed.

PA28αß: the enigmatic magic ring of the proteasome?

PA28αß is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. In cells, PA28 also exists in larger complexes along with the 19S particle, which allows ATP-dependent degradation of proteins; although in vivo a large fraction of PA28 is present as PA28αß-20S particles whose exact biological functions are largely unknown. Although several lines of evidence strongly indicate that PA28αß plays a role in MHC class I antigen presentation, the exact molecular mechanisms of this activity are still poorly understood. Herein, we review current knowledge about the biochemical and biological properties of PA28αß and discuss recent findings concerning its role in modifying the spectrum of proteasome's peptide products, which are important to better understand the molecular mechanisms and biological consequences of PA28αß activity.

Enhanced rate of degradation of basic proteins by 26S immunoproteasomes.

Immunoproteasomes are alternative forms of proteasomes specialized in the generation of MHC class I antigenic peptides and important for efficient cytokine production. We have identified a new biochemical property of 26S immunoproteasomes, namely the ability to hydrolyze basic proteins at greatly increased rates compared to constitutive proteasomes. This enhanced degradative capacity is specific for basic polypeptides, since substrates with a lower content in lysine and arginine residues are hydrolyzed at comparable rates by constitutive and immunoproteasomes. Crucially, selective inhibition of the immunoproteasome tryptic subunit ß2i strongly reduces degradation of basic proteins. Therefore, our data demonstrate the rate limiting function of the proteasomal trypsin-like activity in controlling turnover rates of basic protein substrates and suggest new biological roles for immunoproteasomes in maintaining cellular homeostasis by rapidly removing a potentially harmful excess of free histones that can build up under different pathophysiological conditions.

PA28αß reduces size and increases hydrophilicity of 20S immunoproteasome peptide products.

The specific roles that immunoproteasome variants play in MHC class I antigen presentation are unknown at present. To investigate the biochemical properties of different immunoproteasome forms and unveil the molecular mechanisms of PA28 activity, we performed in vitro degradation of full-length proteins by 20S, 26S, and PA28αß-20S immunoproteasomes and analyzed the spectrum of peptides released. Notably, PA28αß-20S immunoproteasomes hydrolyze proteins at the same low rates as 20S alone, which is in line with PA28, neither stimulating nor preventing entry of unfolded polypeptides into the core particle. Most importantly, binding of PA28αß to 20S greatly reduces the size of proteasomal products and favors the release of specific, more hydrophilic, longer peptides. Hence, PA28αß may either allosterically modify proteasome active sites or act as a selective "smart" sieve that controls the efflux of products from the 20S proteolytic chamber.

The direction of protein entry into the proteasome determines the variety of products and depends on the force needed to unfold its two termini.

Poorly structured domains in proteins enhance their susceptibility to proteasomal degradation. To learn whether the presence of such a domain near either end of a protein determines its direction of entry into the proteasome, directional translocation was enforced on several proteasome substrates. Using archaeal PAN-20S complexes, mammalian 26S proteasomes, and cultured cells, we identified proteins that are degraded exclusively from either the C or N terminus and some showing no directional preference. This property results from interactions of the substrate's termini with the regulatory ATPase and could be predicted based on the calculated relative stabilities of the N and C termini. Surprisingly, the direction of entry into the proteasome affected markedly the spectrum of peptides released and consequently influenced the efficiency of MHC class I presentation. Thus, easily unfolded termini are translocated first, and the direction of translocation influences the peptides generated and presented to the immune system.

Pivotal Advance: Protein synthesis modulates responsiveness of differentiating and malignant plasma cells to proteasome inhibitors.

A previously unsuspected, considerable proportion of newly synthesized polypeptides are hydrolyzed rapidly by proteasomes, possibly competing with endogenous substrates and altering proteostasis. In view of the anti-cancer effects of PIs, we set out to achieve a quantitative assessment of proteasome workload in cells hallmarked by different PI sensitivity, namely, a panel of MM cells, and in a dynamic model of plasma cell differentiation, a process that confers exquisite PI sensitivity. Our results suggest that protein synthesis is a key determinant of proteasomal proteolytic burden and PI sensitivity. In different MM cells and in differentiating plasma cells, the average proteolytic work accomplished per proteasome ranges over different orders of magnitude, an unexpected degree of variability, with increased workload invariably associated to increased PI sensitivity. The unfavorable load-versus-capacity balance found in highly PI-sensitive MM lines is accounted for by a decreased total number of immunoproteasomes/cell coupled to enhanced generation of RDPs. Moreover, indicative of cause-effect relationships, attenuating general protein synthesis by the otherwise toxic agent CHX reduces PI sensitivity in activated B and in MM cells. Our data support the view that in plasma cells protein synthesis contributes to determine PI sensitivity by saturating the proteasomal degradative capacity. Quantitating protein synthesis and proteasome workload may thus prove crucial to design novel negative proteostasis regulators against cancer.