19S Regulator Research Articles

Brain-Derived 11S Regulator (PA28αβ) Promotes Proteasomal Hydrolysis of Elongated Oligoglutamine-Containing Peptides.

Proteins with extended polyglutamine regions are associated with several neurodegenerative disorders, including Huntington's disease. Intracellular proteolytic processing of these proteins is not well understood. In particular, it is unclear whether long polyglutamine fragments resulting from the proteolysis of these proteins can be potentially cleaved by the proteasome. Here, we studied the susceptibility of the glutamine-glutamine bond to proteolysis by the proteasome using oligoglutamine-containing peptides with a fluorophore/quencher pair. We found that the addition of the 11S proteasomal regulator (also known as PA28) significantly accelerated the hydrolysis of oligoglutamine-containing peptides by the 20S proteasome. Unexpectedly, a similar effect was observed for the 26S proteasome in the presence of the 11S regulator. LC/MS data revealed that the hydrolysis of our peptides with both 20S and 26S proteasomes leads to N-terminal fragments containing two or three glutamine residues and that the hydrolysis site does not change after the addition of the 11S regulator. This was confirmed by the docking experiment, which shows that the preferred hydrolysis site is located after the second/third glutamine residue. Inhibitory analysis revealed that trypsin-like specificity is mainly responsible for the proteasomal hydrolysis of the glutamine-glutamine bond. Together, our results indicate that both 20S and 26S proteasomes are capable of degrading the N-terminal part of oligoglutamine fragments, while the 11S regulator significantly accelerates the hydrolysis without changing its specificity. This data suggests that proteasome activity may be enhanced in relation to polyglutamine substrates present in neurons in the early stages of polyglutamine disorders.

Proteomic and Lipidomic Investigation of an Ex-Vivo Human Model of Sickle Cell Disease Erythropoiesis and Patient Erythrocytes Reveals the Abnormal Localization and Dysfunction of the Proteasome, Disrupted Antioxidant Defenses and Altered Lipid Profiles

Introduction: The impact of the early expression of sickle hemoglobin (HbS) on erythroid differentiation remains unclear. We hypothesized that HbS already plays a detrimental role by altering the lipidome/proteome during sickle cell disease (SCD) erythropoiesis. Provided that in-vivo studies do not allow the dynamical follow-through of homogenous populations, are biased by non-cell autonomous interactions, and that bone marrow (BM) harvesting in SCD individuals is difficult to justify, we aimed to compare whole-cell/membrane proteomes and lipidomes in an ex-vivo human model of SCD and healthy erythropoiesis, as well as in mature peripheral blood (PB) erythrocytes. Methods: Healthy and SCD erythropoiesis was modeled ex-vivo in a 3-phase liquid system at ambient O2 tension starting with CD34+ hematopoietic stem cells (HSC) isolated either from healthy donor BM or GSCF-stimulated PB, and SCD patient apheresis products (Panel A). Whole cell protein extracts were obtained at 3 timepoints (day 7, 11, 18, i.e early-, late-erythroblast (EB) and reticulocyte stages). Membrane extracts were purified with a novel erythroid Cell-Surface-Capture method. Proteomes and lipidomes were characterized by quantitative LC-MS/MS and compared according to time-points and health vs. disease states in unbiased/unsupervised models. Multi-Omics Factor Analysis (MOFA) determined key modifiers of SCD erythropoiesis. Moreover, omic analyses were performed on mature healthy and SCD erythrocytes obtained from respective PB (after density-fractionation of light/dense cells for SCD). Proteasome subunits were quantified in healthy vs. SCD erythrocyte ghosts by WB. Proteasome activity was assayed in mature cells and in ex-vivo late-stage SCD erythroid cells under different L-glutamine levels. Results: First, we observed that CD34+ HSCs derived from BM vs. PB generate significantly divergent proteomes/lipidomes in ex-vivo erythropoiesis. Specifically, differences in glutathione and glucose pathway proteins were observed. Amongst them, alpha-enolase is 2-fold to 6-fold depleted in intermediate and reticulocyte stages respectively, when comparing PB vs BM-derived erythropoiesis. This finding is relevant in consideration of prior data on the regulation of HSC erythroid vs myeloid lineage commitment by glucose/glutamine metabolism. Second, we used SCD vs BM-derived erythropoiesis (reference healthy control) to capture the most significant modifications. Among those, the proteasome was progressively abnormally localized in SCD. We observed the depletion of core 20S subunits and sequestration of 19S regulators at the membrane (Panel B), which could be confirmed in dense SCD erythrocyte membranes. Also, we observed an inversion of proteasome activity between membrane and cytosolic extracts in light vs. dense SCD erythrocytes (increased membrane activity in dense SCD erythrocytes). Removing glutamine from culture in the ex-vivo model of SCD erythropoiesis significantly increased the membrane proteasome activity at the reticulocyte stage. Moreover, oxidative stress defenses and cholesterol biosynthesis were perturbed in SCD erythropoiesis, including the upregulation of Heme-oxygenase-1. Also, the antioxidant protein balance was abnormal in mature-dense SCD erythrocyte membranes where insoluble aggregates (i.e. actin/ankyrin + HbS + GAPDH) were sequestered. Additionally, increased GAPDH could be seen in cytoskeleton extracts. Third, we observed the erythroid lipidome to evolve harmoniously in SCD vs healthy erythropoiesis across time-points, while significant perturbations were seen once erythrocytes are in circulation. In fact, dense SCD erythrocytes had significantly abnormal levels of lysophosphatidylcholines (depleted), sphingomyelins (depleted) and triacylglycerols (enriched). Fourth, MOFA revealed the key contribution of heme synthesis, glycolysis and proteasome regulation to the differences in global phenotype between SCD and healthy erythropoiesis. Conclusion: Our study shows that a) ex-vivo human erythropoiesis models based on healthy PB vs BM-derived HSCs differ significantly on a lipidomic/proteomic level, highlighting the importance of source choice, b) HbS expression has an incremental proteomic impact during erythropoiesis and c) the lipidome is unaltered in SCD erythropoiesis but radically changes once erythrocytes are circulating. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Essential Drug Targets and Pathways in PI-Resistant MM Cells

BackgroundProteasome inhibitors (PI) have emerged as a powerful, cell biology-based treatment option for multiple myeloma (MM) and build a central backbone for MM treatment with three proteasome-inhibiting drugs currently approved: bortezomib (BTZ), carfilzomib (CFZ) and ixazomib. However, despite the high anti-MM activity of PI, MM cells adapt to the selective pressure of PI treatment in most cases to date and most MM patients relapse during or after treatment with PI, develop PI-refractory disease and ultimately die. Therefore, understanding and overcoming PI resistance is a key challenge for MM therapy. Our previous in vitro studies on PI-resistant MM suggest that PI-adapted, MM cells show very distinct features of general metabolism and cell biology that differentiate them from PI-sensitive MM, derived from the same cell line. We hypothesize that this highly specialized and adapted nature of PI-resistant MM offers novel areas of vulnerability, that differ from the therapeutic targets in PI-sensitive MM. The aim of our study was to identify essential drug targets and pathways in PI-resistant MM using genome-wide functional screening with the CRISPR/Cas9 system that could serve as novel therapeutic targets in PI-resistant MM.MethodsWe used genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library in L363-BTZ and RPMI-8226-BTZ cells, adapted to grow in the presence of 90 nM BTZ. The overlapping bortezomib genetic sensitivity candidates were further validated in the set of BTZ-resistant cells (L363-BTZ, RPMI-8226-BTZ, MM1S-BTZ and AMO-BTZ) cells using shRNA silencing or single-gene specific knockout or genetic overexpression using CCK8 viability assay. Subsequent functional analysis of the highest ranking BTZ sensitivity candidates in BTZ-adapted cells included apoptosis and cell cycle analysis, qPCR and western blotting, SILAC, proteasome activity determination using activity-based probes and FRAP analysis.ResultsCRISPR/Cas9-screening identified two candidate genes for BTZ sensitivity, ECPAS (KIAA0368; Ecm29 Proteasome Adaptor and Scaffold protein) and PSME1 (an 11S regulator complex subunit), as consistent screening hits in two independent BTZ-adapted MM cell lines. Both genes are related to proteasome, but do not build the proteasome core particle and do not have a proteolytic activity. Specific knock-down or knock-out of ECPAS sensitized PI-naïve cells to BTZ and CFZ, while significantly more sensitizing BTZ-adapted cells to both PI. Likewise, overexpression of PSMF1, an inhibitor of 11S regulator complex, sensitized BTZ-resistant as well as sensitive cells to BTZ. ECPAS-depleted BTZ-adapted cells showed accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, cell cycle arrest and induction of apoptosis, together with changes in protein synthesis after the treatment with 50 nM bortezomib, in contrast to BTZ-adapted control cells. FRAP analysis of cells with GFP-tagged PSMD6 revealed that the intracellular mobility of proteasomes in ECPAS-depleted cells was reduced. Importantly, proteasome activity determined by activity-based probes was not impaired in ECPAS-depleted cells.ConclusionIn conclusion, BTZ-resistant MM cells uniquely show a high dependency on the proteasome adaptor and scaffold protein ECPAS, which has been shown to be involved in coupling of proteasome in different compartments and promotes proteasome dissociation under oxidative stress. Specifically in PI-resistant MM, ECPAS is important to ensure functional proteasome, is involved in controlling the intracellular mobility of proteasomes, likely to ensure high proteasome turnover. ECPAS therefore represents a novel candidate that may be targeted to specifically re-sensitize PI-resistant MM cells to proteasome inhibitor treatment. DisclosuresNo relevant conflicts of interest to declare.

Abstract A47: The immunoproteosome mediates resistance to natural killer cell killing

Abstract We identified the immunoproteosome as a mediator of resistance to natural killer (NK) cell killing through a genome-wide activation CRISPR screen. The immunoproteosome is a modified proteolytic complex composed of a catalytic 20S core and a regulatory 11S component. An essential function of the immunoproteosome is to degrade intracellular proteins that are eventually presented as antigens on the cell surface by the major histocompatibility complex (MHC) I. We validated several subunits that play a critical role in regulating responses to NK cells, specifically, PSMA1, PSMB6 and PSME1, which encode the alpha subunit of the 20S catalytic proteosome, the beta subunit of the 20S proteosome, and the alpha subunit of the 11S regulator, respectively. While several studies have reported that proteasome inhibitors sensitize cancer cells to NK cell killing, the exact mechanisms of these inhibitors are not well understood. Our data indicate that these proteasome components may be attractive targets to improve cancer cell response to NK cell-based therapies. Note: This abstract was not presented at the conference. Citation Format: Joyce Pasion, Hans Guido Wendel. The immunoproteosome mediates resistance to natural killer cell killing [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A47.

PSMD12 promotes breast cancer growth via inhibiting the expression of pro-apoptotic genes

Breast cancer (BC), the most frequent cancer in women worldwide, is extremely heterogeneous. For effective and precise treatment and to cope with drug resistance in BC, we need to find more therapeutic molecular targets. In this study, we found that the Proteasome 26S Subunit, Non-ATPase 12 (PSMD12) was upregulated in BC samples, its expression was heterogeneous among different cell lines, and high levels of PSMD12 were related to poor prognosis of BC patients. Notably, the expression of PSMD12 increased in the nucleus. Cytological experiments revealed that PSMD12 knockdown inhibited cell growth and migration, and a genome-wide CRISPR-Cas9 knockout (GeCKO) screen also confirmed that PSMD12 is a crucial gene for the growth of BC cells. Flow cytometry showed that cell apoptosis increased in the PSMD12 knockdown, and RNA-seq indicated that the apoptosis pathway was activated, and the TXNIP, GADD45A, GADD45B, RHOB, and CDKN1A pro-apoptotic genes were highly expressed, a result that was validated by RT-qPCR and Western blot. Furthermore, restoration of PSMD12 expression decreased the expression of pro-apoptotic genes. A tumor-bearing mice assay demonstrated that BC growth was arrested by reduced PSMD12 levels in vivo. Taken together, PSMD12, a subunit of 19S regulator of 26S proteasome, was identified as a potential prognostic and therapeutic molecular target for BC, which provides a new insight for developing anticancer drugs that promote apoptosis based on the targeting of the 26S proteasome complex.

Expression of Concern: Reduction in ATP Levels Triggers Immunoproteasome Activation by the 11S (PA28) Regulator during Early Antiviral Response Mediated by IFNβ in Mouse Pancreatic β-Cells.

Expression of Concern: Reduction in ATP Levels Triggers Immunoproteasome Activation by the 11S (PA28) Regulator during Early Antiviral Response Mediated by IFNβ in Mouse Pancreatic β-Cells.

Expanding the Clinical and Molecular Spectrum of PSMD12-Related Neurodevelopmental Syndrome: An Additional Patient and Review

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process that eukaryotic cells use to regulate their functions and coordinate their signaling networks. In particular, it plays a crucial role in neuronal development regulating very important functional and morphological interplays of neurons, such as synaptic plasticity, neurotransmitter release and morphogenesis of axons. Pathogenic alterations of genes involved in the proteolysis by UPS have been associated with several neurodevelopmental disorders (NDDs) and human diseases, highlighting the importance of this regulatory mechanism to developmental processes and neurogenesis. Here, we describe a 19 years old male patient showing a syndromic form of NDD. The main clinical features are intellectual disability/speech delay, congenital anomalies and facial dysmorphisms. Through a targeted resequencing approach (TRS), we identified a missense variant in PSMD12, a gene recently associated to an emerging syndromic form of NDD, which encodes for the non-ATPase subunit of the 19S regulator of 26S proteasome complex. The variant described herein, inherited from the father with apparently borderline cognitive ability, is useful to expand the molecular spectrum of heterozygous PSMD12 mutations and to provide insight into the molecular pathogenesis of this new condition since it is, to the best of our knowledge, the first missense substitution to date reported in medical literature. More importantly, our study highlight once again the utility of next generation sequencing in establishing an etiological basis in clinically and genetically heterogeneous conditions such as NDDs, thus allowing a better diagnosis, counseling and management of affected patients and their families.

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

PA28γ 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, PA28γ 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 PA28γ, 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 PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.

Protein Homeostasis: A Key Target in Myeloma

Multiple myeloma is a disease of long-lived plasma cells that remains incurable in the majority of patients. However advances in the treatment have been made in the last 20 years resulting in significant improvement in the survival of patients with this disease. Interestingly almost all of these advances have been made through the targeting of pathways or molecules that are shared between normal and malignant plasma cells and are not due to the precision targeting of myeloma cell dependencies associated with the transformation process. Most notably targeting both specific and bulk protein homeostasis through the action of IMiDs and proteasome inhibitors have become the backbone for the treatment of newly diagnosed myeloma in the United States. We have focused on proteasome function and regulation to better understand why a macromolecular structure that is present and functions in every cell in the body is such an effective target for myeloma therapy. Our initial findings indicated that proteasome inhibition activated an endoplasmic reticulum stress response known as the unfolded protein response (UPR). The UPR is required for normal differentiation and maintenance of plasma cells, although our initial findings suggesting that the PERK arm of the response, which is not required for normal plasma cell maintenance was activated by proteasome inhibitors. We determined that the likely source of unfolded protein was immunoglobulin produced by myeloma cells and that the fate of immunoglobulin was a key determinant of sensitivity to proteasome inhibition. We have now focused on additional factors that determine the response to proteasome inhibition and consistent with previous findings have demonstrated that activation of heat shock response is a early protective event following proteasome inhibition. However we were unable to demonstrate that any single heat shock protein was sufficient to alter the response to proteasome inhibition. Rather it was the activation of the heat shock response itself and that protection was only efficiently blocked by inhibition of the transcriptional regulator of the response, HSF1. HSF1 is activated through post-translational modifications and proteasome inhibition results in phosphorylation of HSF1 at several sites, most notably S326. This can be blocked with the multi-kinase inhibitor TG02 that effectively blocks the proteasome inhibitor-induced heat shock response and sensitizes myeloma cells to proteasome inhibitor-induced cell death. Based on these findings, the combination of TG02 and carfilzomib are being tested clinically. The cellular response to proteasome inhibitors is also controlled through the regulation of proteasome assembly. We have recently demonstrated that 14-3-3ζ an anti-apoptotic phospho-serine binding protein, can limit proteasome assembly and activity through its binding to the PA28α subunit of the 11S regulator. This significantly lowers proteasome capacity and sensitizes cells to proteasome inhibition but not to other therapeutic agents. Finally in addition to regulation of bulk protein homeostasis, targeting of specific protein turnover has proven effective in myeloma treatment. IMiDs can alter the ability of the E3 ligase cereblon to recognize substrates for ubiquitination, resulting in the degradation of key B cell transcription factors IKZF1/3. These can regulate the plasma cell maintenance factor IRF4 as well as play a direct role in the regulation plasma cell superenhancers that control the expression of oncogenes translocated in myeloma. Another key mediator of myeloma cell survival, MCL1 is regulated via protein stability and can targeted in this manner. Direct and indirect targeting of MCL1 will be discussed. Disclosures Boise: Abbvie: Consultancy; Eli Lilly and Company: Research Funding.

De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

194 - Measuring 20S Proteasome and Immunoproteasome Activities in Oxidative Stress

Protein degradation mechanisms are fundamental components in maintaining proteostasis and homeostasis. Intracellular proteins are highly susceptible to oxidative modification and damage. Inactivation of key enzymes and aggregation or cross-linking of numerous cell proteins are only some of the known outcomes of intracellular protein oxidation. Ultimately, protein oxidation, modification, aggregation, and cross-linking have been linked to multiple diseases and to the aging process. Our work has shown that oxidized proteins in the cytoplasm, nucleus, and endoplasmic reticulum are selectively degraded by the 20S Proteasome and the Immunoproteasome (both ± the 11S or Pa28 proteasome regulator) whereas oxidatively damaged mitochondrial proteins are degraded by the Lon protease. Rapid degradation of mildly damaged proteins prevents their aggregation and cross-linking, and allows for their replacement by de novo synthesis. Increased transcription/translation of key proteolytic enzymes in response to signaling levels of oxidants is a key feature of Adaptive Homeostasis. In the past few years, it has become commonplace to evaluate discrete proteasome activities using the cleavage of small fluorogenic peptides to measure caspase-like activity, trypsin-like activity, and chymotrypsin-like activity. Our studies demonstrate that substrate concentration, cell type or proteolytic enzyme concentration, and length of incubation are all key factors in determining the proteolysis of fluorogenic peptides. Our work provides a rational and quantitative approach to the measurement of proteasomal activities under both normal and stress conditions. This work has been supported by grant ES003598 from the National Institute of Environmental Health Sciences of the US National Institutes of Health to K.J.A.D.

Amyloid-β Increases Activity of Proteasomes Capped with 19S and 11S Regulators.

Accumulation of amyloid-β (Aβ) in neurons accompanies Alzheimer's disease progression. In the cytoplasm Aβ influences activity of proteasomes, the multisubunit protein complexes that hydrolyze the majority of intracellular proteins. However, the manner in which Aβ affects the proteolytic activity of proteasomes has not been established. In this study the effect of Aβ42 and Aβ42 with isomerized Asp7 on activity of different forms of proteasomes has been analyzed. It has been shown that Aβ peptides efficiently reduce activity of the 20S proteasomes, but increase activity of the 20S proteasomes capped with the 19S and/or 11S regulators. Modulation of proteasome activity is mainly determined by the C-terminal segment of Aβ (amino acids 17-42). This study demonstrated an important role of proteasome regulators in the interplay between Aβ and the proteasomes.

The 11S proteasome activator: Isolation from mouse brain and the influence on peptide substrate hydrolysis of the 20S and 26S proteasomes

Purification of the 11S proteasome regulator from mouse brain was optimized; the subunit composition of the isolated protein was determined by Western blot. The dependency of 20S proteasome peptidase activity on the molar concentration of the 11S regulator was examined. The Michaelis constants of hydrolysis of the specific fluorescent substrates Suc–Leu–Leu–Val–Tyr–AMC, Ac–Arg–Leu–Arg–AMC, and Z–Leu–Leu–Glu–AMC by the 20S proteasome from BALB/c mouse brain and the 20S–11S complex were determined. It was shown that the 11S particle has almost no influence on binding of specific fluorescent substrates to the 20S proteasome, but strongly accelerates hydrolysis of all three substrates, while not affecting the rate of peptide substrate hydrolysis by the 26S proteasome.

Surrogate Prognostic Biomarkers in OSCC: The Paradigm of PA28γ Overexpression

Carcinomas of the head and neck represent the sixth most commonly diagnosed type of cancer. Most of head and neck carcinomas are histologically categorized as squamous cell carcinomas (SCC) (Chin et al., 2006). SCC arises from squamous cells lining mucosal surfaces in the nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, oral cavity, oropharynx, salivary glands, and ears (Argiris et al., 2008). The most common type of head and neck SCC is oral squamous cell carcinoma (OSCC), with an estimated annual incidence of 199 thousands of cases and an estimated mortality of 98 thousands, only in men, as the incidence of OSCC is much higher in men than in women (Bray et al., 2012). The causative risk factors of OSCC include tobacco use and alcohol consumption (Argiris et al., 2008). A wide gamut of molecules is involved in oral carcinogenesis. Aberrant expression of several genes is associated with hallmarks of OSCC, including uncontrolled cell proliferation, defective apoptosis, loss of cell differentiation, epithelial–mesenchymal transition, metastasis, and angiogenesis (da Silva et al., 2011). The potential of such molecules as molecular biomarkers for diagnosis, prognosis or monitoring of treatment efficacy in OSCC has been intensively investigated during the last decades. Immunohistochemical detection and/or mRNA expression profiling of promising molecular biomarkers are expected to significantly contribute to the generation of novel screening tests with high sensitivity and specificity, as well as tailor-made therapies against OSCC, in the era of personalized medicine that has just emerged (Cheng et al., 2014). Besides that, selective targeting of interactions between specific molecules that are key players in cancer-related biochemical pathways could dramatically impede oral carcinogenesis and therefore elongate or ameliorate the survival of OSCC patients. The proteasome activator 28 gamma (PA28γ) encodes the gamma subunit of the 11S regulator of the immunoproteasome, a modified proteasome that processes class I MHC peptides. PA28γ is a multifunctional protein, implicated in the degradation of important cell cycle regulatory proteins in an ATP-independent manner. In fact, PA28γ inhibits apoptosis and promotes cell cycle progression (Rechsteiner and Hill, 2005). A homopolymer form of PA28γ interacts with both MDM2 and p53 proteins and facilitates their physical interaction, which promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, thus limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28γ in human cancer cells has been shown to abolish MDM2-mediated p53 degradation, increase the activity of p53, and enhance apoptosis (Zhang and Zhang, 2008). In the E-Biomedicine, Jing Li and colleagues Li et al., 2015 assess immunohistochemically PA28γ expression as a prognostic biomarker in OSCC. For this purpose, a total of 368 patients from three independent cohorts were included in the current study. Moreover, the authors used an independent cohort of 460 patient specimens obtained between 1992 and 2013 in the TCGA database as an external validation cohort to validate the prognostic value of PA28γ expression. Strong PA28γ expression was shown to predict significantly reduced disease-free and overall survival in OSCC patients. More importantly, the unfavorable prognostic value of PA28γ was independent from other prognostic factors such as smoking history, drinking history, cell differentiation, tumor stage, nodal stage, radiotherapy and chemotherapy. Besides the prognostic significance of PA28γ in OSCC, this study provides evidence for the role of PA28γ in oral tumor growth and metastasis. In more detail, PA28γ silencing in two OSCC cell lines led to a decline in cell viability and colony growth. Moreover, growth of tumor originating from OSCC cells transplanted subcutaneously on the right back of BALB/c nude mice was much slower when PA28γ was silenced in these tumor cells. The authors also showed that PA28γ silencing suppresses tumor angiogenesis and increases tumor cell apoptosis in xenograft models. This study opens the door to assess the clinical value of PA28γ expression as a surrogate prognostic biomarker in larger cohorts of OSCC patients as well as to further investigate the role of PA28γ overexpression in the pathobiology and progression of OSCC. The clinical value of several molecules as putative diagnostic, prognostic, and treatment-response biomarkers in OSCC has been widely investigated during the last decades (Chawla et al., 2015, Ni et al., 2015). The elucidation of the biochemical pathways implicated in oral carcinogenesis, tumor progression, and metastasis, taking also into consideration the heterogeneity of oral tumors, would definitely assist the discovery of novel candidate biomarkers in OSCC. Some molecular biomarkers, including PA28γ, may also have prognostic value in specific OSCC patient subgroups. In conclusion, the dysregulation of PA28γ gene expression in OSCC tissues compared to benign oral tumors, dysplasias, or normal specimens originating from the oral cavity merits further investigation and validation in independent and large cohorts of OSCC patients.

Proteasome subtypes and regulators in the processing of antigenic peptides presented by class I molecules of the major histocompatibility complex.

The proteasome is responsible for the breakdown of cellular proteins. Proteins targeted for degradation are allowed inside the proteasome particle, where they are cleaved into small peptides and released in the cytosol to be degraded into amino acids. In vertebrates, some of these peptides escape degradation in the cytosol, are loaded onto class I molecules of the major histocompatibility complex (MHC) and displayed at the cell surface for scrutiny by the immune system. The proteasome therefore plays a key role for the immune system: it provides a continued sampling of intracellular proteins, so that CD8-positive T-lymphocytes can kill cells expressing viral or tumoral proteins. Consequently, the repertoire of peptides displayed by MHC class I molecules at the cell surface depends on proteasome activity, which may vary according to the presence of proteasome subtypes and regulators. Besides standard proteasomes, cells may contain immunoproteasomes, intermediate proteasomes and thymoproteasomes. Cells may also contain regulators of proteasome activity, such as the 19S, PA28 and PA200 regulators. Here, we review the effects of these proteasome subtypes and regulators on the production of antigenic peptides. We also discuss an unexpected function of the proteasome discovered through the study of antigenic peptides: its ability to splice peptides.

Signaling control of proteasome, immunoproteasome, and PA28 regulator gene expression during adaptation to oxidative stress (555.11)

Levels of oxidative stress change continuously, depending on environment, metabolism, foods, drugs and medications, and radiation. Young, healthy, mammalian cells can readily adapt to increases in oxidative stress such that they become (temporarily) much more resistant to oxidative damage. Intracellular proteins are major targets of oxidative stress and it is vitally important that cells rapidly and selectively degrade mildly oxidized proteins, before they undergo more severe oxidation, aggregation, and cross‐linking. Our studies show that such adaptive responses include the immediate disassembly of the 26S proteasome (catalyzed by HSP70 and Ecm‐29) to form free 20S proteasome and 19S regulator complexes, at which point ATP/Ubiquitin‐dependent proteolysis is temporarily lost. The additional free 20S Proteasomes (+/‐ 11S or PA28 regulators) are of immediate help in degrading oxidized proteins. The original 26S Proteasomes are re‐assembled, and ATP/Ubiquitin‐dependent proteolysis is restored over a three‐hour period. During this three‐hour period, and during the subsequent 17 hours, new 20S Proteasomes, Immunoproteasomes, and 11S (PA28) proteasome regulators are synthesized, partially under the control of the Nrf2 signal transduction pathway. We have now obtained similar findings in C. elegans worms, and D. melanogaster fruit flies. Proteasome and Immunoproteasome subunit genes, and the 11S (PA28) regulator appear to be true shock/stress genes that provide significant oxidative stress protection. In older cells, Proteasome activities decline, and adaptational responses mediated by Nrf2 become sluggish or even ineffectual. The antagonistic effects of Bach1 and/or Nrf1 may actually blunt adaptive proteolytic responses to stress in older cells and organisms. Studies in animals and humans suggest that declining Proteasome activities and, perhaps, declining responsiveness to stress, may contribute to the ageing process, and to various age‐associated diseases.Grant Funding Source: NIH/NIEHS ES003598

Proteasomes on thyroid tissue allotransplantation under induction of donor specific tolerance in rats

The proteasomes in the liver of August rats (RT1C) were investigated 30 days after the allotransplantation of Wistar rat (RT1u) thyroid tissue under renal capsule with/without induction of donor specific tolerance by donor splenocyte intraportal administration. The level of the total proteasome pool, immune proteasomes containing the LMP2 and/or LMP7 subunits, proteasome 19S- and 11S-regulators was defined. The intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups that indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of non-tolerated rats compared to tolerated animals. In the liver of tolerated rats with survived transplants, the quantity of mononuclear cells, expressing the immune subunit LMP2, greatly increased in comparison with control and non-tolerated animals. Study of the survived transplants showed the increase of the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them compared to the tissue replacing the rejected transplants. In the control intact thyroid tissue, the immune proteasomes were almost not revealed, while 19S/11S ratio was maximal. Thus, the development of the immune reaction or its suppression is accompanied by change of the balance between different proteasome forms. The immune subunit LMP7 and 11S regulator are connected with the response against donor tissue. On the contrary, the immune subunit LMP2 and 19S regulator are likely to be important for the immune tolerance development and survived tissue functioning. The low content of the immune proteasomes in the follicle cells was found by immunofluorescence assay. The formation of antigens for major histocompatibility complex class I molecules was impaired by low immune proteasome content that led to immunological tolerance to hormone-producing follicle cells.

Regulation of ubiquitin and 26S proteasome mediated by phenolic compounds during oxidative stress

Little attention has been devoted to studying the roles of natural antioxidants in the ubiquitin-proteasome pathway during oxidative stress. We demonstrated that a time course revealed that the reassociation of the 19S regulators with the 20S proteasomes occurred automatically and rapidly to reconstitute the 26S proteasomes, with up to 80% completion, within 5 min after H2O2 treatment. Ubiquitin, methyl gallate and tannic acid are able to prevent H2O2 from inhibiting the 26S activity. We further show that the level of the ubiquitin, S5a and 20S core subunits decreased within 30 min and increased after 24 h of H2O2 treatment in Hep-2 cells. Phenolic compounds not only inhibited the 26S activity but also decreased the USP47 levels, which reduce the DNA damage repair rate during oxidative stress; in addition, the presence of DNA fragments, procaspase-3 and a decreased poly (ADP-ribose) polymerase also appeared as a result of the above conditions. Ubiquitin could serve as a protective substrate in H2O2 and phenolic compound-treated Hep-2 cells. Methyl gallate and tannic acid, as prooxidants, can attenuate the apoptotic response resulting from long-term oxidative stress. Collectively, these data demonstrate an important role for phenolic compounds in regulating the 26S proteasome and ubiquitin during oxidative stress.

Reduction in ATP Levels Triggers Immunoproteasome Activation by the 11S (PA28) Regulator during Early Antiviral Response Mediated by IFNβ in Mouse Pancreatic β-Cells

Autoimmune destruction of insulin producing pancreatic β-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic β-cells. Of special interest to immunoproteasome activation in β-cells are the effects of IFNβ, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNγ, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic β1i, β2i and β5i, and 11S mRNAs upon exposure to IFNβ or IFNγ. Higher concentrations of IFNβ than IFNγ are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNβ-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic β-cells exposed to IFNβ, but primarily in cells with reduced ATP levels that stimulate the 11S participation in immunoproteasome function.

Effects of Cardiomyopathy-Related Troponin T Mutations on the Ubiquitin-Proteasome System

Familial hypertrophic cardiomyopathy (FHC) is a disease of the myocardium that can be caused by a mutation in a sarcomeric gene. TNNT2 encodes cardiac troponin T (cTnT), a sarcomeric protein important for cardiac muscle contraction. Mutations in TNNT2 are frequently linked to sudden cardiac death (SCD); however their role in heart failure and cardiomyopathy is unclear. We hypothesize that changes in the ubiquitin-proteasome system (UPS) are related to increased risk of cardiac death, and that changes in the UPS may be related to calcium sensitization caused by TNNT2 mutations. We examined two mutations in TNNT2, R278C, which is associated with mild effects and late-onset heart disease, and the calcium-sensitizing mutation I79N, which is early-onset and has severe cardiac effects, including SCD. In 3-month-old transgenic mice expressing the I79N mutant form of troponin T, expression of the proteasome subunit PSMA6 was decreased by approximately 35% in I79N mice when compared to hearts from wild-type transgenic mice. Protein levels of the 19S regulator subunit Rpt1 were increased in the hearts of 3-month-old I79N mice and decreased in the hearts of 1-year-old R278C mice. Expression of PKC alpha, an important regulator of cardiac contractility, was decreased in R278C mice at 1 year. Activity of the lysosomal protease cathepsin L was increased in 1-year-old I79N mice, indicating disruption of another major proteolytic system in these hearts. Quantitative real-time PCR of the genes involved in the ubiquitination pathway (E1, E2, and E3 enzymes) showed that transcript levels were increased or decreased for 25 genes in I79N mice and three genes in R278C mice. These data demonstrate that the UPS is affected at the gene expression and protein level in R278C and I79N mice, indicating that changes in the UPS may be involved in FHC.