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The proteasome has been validated as an anticancer drug target, while the role of a subunit of proteasome, PSMC6, in lung adenocarcinoma (LUAD) has not been fully unveiled. In this study, we observed that both the RNA and protein of PSMC6 were highly upregulated in LUAD compared with the adjacent normal tissues. Moreover, a high PSMC6 expression was associated with poor prognosis. In accordance with this finding, PSMC6 was associated with poor tumor differentiation. Furthermore, the silence of PSMC6 by small interference RNAs (siRNAs) could significantly inhibit cell growth, migration, and invasion in lung cancer cell lines, suggesting that PSMC6 might serve as a promising therapeutic target in LUAD. To further explore the molecular mechanism of PSMC6 in LUAD, we observed that the proteasome subunits, such as PSMD10, PSMD6, PSMD9, PSMD13, PSMB3, PSMB1, PSMA4, PSMC1, PSMC2, PSMD7, and PSMD14, were highly correlated with PSMC6 expression. Based on the gene set enrichment analysis, we observed that these proteasome subunits were involved in the degradation of AXIN protein. The correlation analysis revealed that the positively correlated genes with PSMC6 were highly enriched in WNT signaling-related pathways, demonstrating that the PSMC6 overexpression may activate WNT signaling via degrading the AXIN protein, thereby promoting tumor progression. In summary, we systematically evaluated the differential expression levels and prognostic values of PSMC6 and predicted its biological function in LUAD, which suggested that PSMC6 might act as a promising therapeutic target in LUAD.

Three unrelated patients with similar microdeletions of chromosome 14q32.11 with shared phenotypes including language and developmental delay, and four overlapping genes -CALM1, TTC7B, PSMC1, and RPS6KA5 have been presented. All four genes are expressed in the brain and have haploinsufficiency scores, which reflect low tolerance to loss of function variation. An insight on the genes in the overlapping region, which may influence the resulting phenotype has been provided. Given the three patients' similar phenotypes and lack of normal variation in this region, it was suggested that this microdeletion may be associated with developmental and language delay.

Heterotopic ossification (HO) is frequently seen in patients with spinal injuries. Therefore, this study aimed to characterize the association of HO with ankylosing spondylitis (AS) through gene expression profiling. The human transcriptomic datasets (GSE73754 and GSE94683) were obtained from the Gene Expression Omnibus database for analysis. Overlapping differentially expressed genes (DEGs) were identified between AS and HO disease states. Subsequently, weighted gene co-expression network analysis (WGCNA) was performed for constructing and identifying hub genes for each condition. Finally, a consensus of the overlapping DEGs and the hub genes in AS and HO was taken for determining the key genes involved in AS-induced HO. Quantitative real-time polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels in mesenchymal stem cells of AS patients and controls. Additionally, immunohistochemistry was performed on interspinous ligament samples for experimental validation of genes. DEG analysis identified 355 overlapping genes between HO and AS. WGCNA indicated that the salmon module of the 22 modules constructed, was most significantly correlated with AS-induced HO. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of the salmon module indicated the presence of genes enriched in proteasome regulatory particle and proteasome pathways. mRNA expression analysis identified TCP1 and PSMC1 as the key genes in AS-induced HO. Further validation of these genes could help elucidate their role in the complex association of AS and HO.

African swine fever virus (ASFV) is prevalent in many countries and is a contagious and lethal virus that infects pigs, posing a threat to the global pig industry and public health. The interaction between the virus and the host is key to unlocking the mystery behind viral pathogenesis. A comprehensive understanding of the viral and host protein interaction may provide clues for developing new antiviral strategies. Here, we show a network of ASFV MGF360-9L protein interactions in porcine kidney (PK-15) cells. Overall, 268 proteins that interact with MGF360-9L are identified using immunoprecipitation and liquid chromatography–mass spectrometry (LC-MS). Accordingly, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted, and the protein–protein interaction (PPI) network was created. It was speculated that the cellular proteins interacting with MGF360-9L are involved in protein binding, metabolism, and the innate immune response. Proteasome subunit alpha type (PSMA3), 26S protease regulatory subunit 4 (PSMC1), autophagy and beclin 1 regulator 1 (AMBRA1), and DEAD-box helicase 20 (DDX20) could interact with MGF360-9L in vitro. PSMA3 and PSMC1 overexpression significantly promoted ASFV replication, and MGF360-9L maintained the transcriptional level of PSMA3 and PSMC1. Here, we show the interaction between ASFV MGF360-9L and cellular proteins and elucidate the virus–host interaction network, which effectively provides useful protein-related information that can enable further study of the potential mechanism and pathogenesis of ASFV infection.

Thermal and saline variations of the Southern Ocean are important signs of climate change which can alter the physiological responses of stenotic species residing at high latitudes. Our study aimed to evaluate the cellular stress response (CSR) of Harpagifer antarcticus subjected to increased ambient temperature and decreased salinity. The fish were distributed in different thermal (2, 5, 8, 11, and 14 °C) and saline (23, 28, and 33 psu) combinations for 10 days. We used qPCR analysis to evaluate the transcription of genes involved in the thermal shock response (HSP70, HSC70, HSP90, and GRP78), ubiquitination (E2, E3, ubiquitin, and CHIP), 26S proteasome complex (PSMA2, PSMB7, and PSMC1), and apoptosis (SMAC/Diablo and BAX) in the liver and gill. The expression profiles were tissue-specific and mainly dependent on temperature rather than salinity in the gill; meanwhile, in the liver, both conditions modulated the expression of these genes. Transcription of markers involved in the heat shock response was much higher in the liver than in the gill and was higher when salinity decreased and the temperature increased. Similarly, the genes involved in the ubiquitination pathway, 26S complex of the proteasome, and the apoptotic pathway showed the same pattern, being mainly induced in the liver rather than in the gill. This is the first study to show that this Antarctic fish can induce the cellular stress response in their tissues when subjected to these thermal/saline combinations.

Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 “resistance” genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862–70. ©2017 AACR.

RationaleThe ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP) are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime for example, show cardiotoxicity. Moreover, UPS and ALP defects are implicated in the pathogenesis of a large subset of heart disease. Hence, a better understanding of the cross-talk between the two catabolic pathways should help advance cardiac pathophysiology and medicine.ObjectiveSystemic pharmacological proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. The present study investigates whether cardiomyocyte-restricted PSMI activates myocardial ALP and, more importantly, how proteasome malfunction activates the ALP in the heart.Methods and ResultsMyocardial macroautophagy, transcription factor EB (TFEB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation ofPsmc1(a 19S proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted markedly by calcineurin inhibition (cyclosporine A) and by siRNA-mediatedMolcn1silence. PSMI induced remarkable increases in myocardial autophagic flux in wild type mice but not p62 null mice. In cultured wild type, but not p62-null, mouse cardiomyocytes, PSMI induced increases in LC3-II flux and in the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62 null mice compared with wild type mice.Conclusions(1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) both Mocln1 and p62 form a feed-forward loop with TFEB during TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac ALP activation in a proteasome malfunction setting.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated β-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect β-catenin, demonstrating USP14 but not UCHL5 has a specific effect on β-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides β-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

Breast cancer is a complex disease, and several processes are involved in its development. Therefore, potential therapeutic targets need to be discovered for these patients. Proteasome 26S subunit, ATPase gene (PSMC) family members are well reported to be involved in protein degradation. However, their roles in breast cancer are still unknown and need to be comprehensively researched. Leveraging publicly available databases, such as cBioPortal and Oncomine, for high-throughput transcriptomic profiling to provide evidence-based targets for breast cancer is a rapid and robust approach. By integrating the aforementioned databases with the Kaplan–Meier plotter database, we investigated potential roles of six PSMC family members in breast cancer at the messenger RNA level and their correlations with patient survival. The present findings showed significantly higher expression profiles of PSMC2, PSMC3, PSMC4, PSMC5, and PSMC6 in breast cancer compared to normal breast tissues. Besides, positive correlations were also revealed between PSMC family genes and ubiquinone metabolism, cell cycle, and cytoskeletal remodeling. Meanwhile, we discovered that high levels of PSMC1, PSMC3, PSMC4, PSMC5, and PSMC6 transcripts were positively correlated with poor survival, which likely shows their importance in breast cancer development. Collectively, PSMC family members have the potential to be novel and essential prognostic biomarkers for breast cancer development.

Introduction: Hypertension occurs profoundly in the world, and left ventricular (LV) remodeling containing functional, structural, and mechanical changes induced by uncontrolled blood pressure is a well-known complication, however the underlying mechanism is still obscure. Methods: To determine differences in gene expression profiles of hypertension and LV remodeling consequence to hypertension, Gene Expression Omnibus 2R online tool was used to identify differently expressed genes. Publicly available databases including GeneMANIA, database for annotation, visualization and integrated discovery, search tool for the retrieva predicting associated transcription factors (TF) from annotated affinities interacting genes, Predicting Associated TF from Annotated Affinities, JASPAR and Comparative Toxicogenomics Database (CTD) were accessed to perform an integrated bioinformatic analysis. Results: Twenty-one genes (SEC14L3, EML7, PSMD7, PSMA1, GLRX, CNOT10, NBR1, DUSP12, STRAP, SMIM14, RBM8A, TMEM59, TMEM87A,PSMC1, CASP4, ITGB8, DNAJA1, PINK1, PRNP, SAP30L, and EIF3M) were found overexpression in both hypertension and hypertensive LV remodeling. Biological process analysis first revealed that enrichment of these target genes correlated with regulation of cellular amino acid metabolic process, antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent and proteasome complex, 3 different expression genes (DEGs) participate significantly enriched in NFκB, WNT, and MAPK pathways, meanwhile, 47% DEGs displayed similar co-expression characteristics. Furthermore, the transcription factors associated with key DEGs were identified. Finally, the TF (HAND1, E4BP4, ESR1, VBP, ELK-1, POU3F2) associated with LV remodeling in hypertension were confirmed to act a crucial role in correlated heart diseases. Conclusion: The present study reveals the targeted genes probably associated with LV remodeling in hypertension by bioinformatics-based analyses, which provides clues for prognosis judgement and pharmacological therapies.

The exact cause instigating multiple myeloma (MM) has not been fully elucidated, and the disease has a median survival of 6 months without any treatment. To identify potential biomarkers of MM, serum proteins reflecting alteration in their proteomes were analyzed in 6 patients with MM compared with 6 healthy controls using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of flight mass spectrometry. The most notable differentially expressed proteins were validated by immunoblotting and changes in mRNA expression were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A total of 11 differentially expressed protein spots were found. The expression levels of 7 proteins [Immunoglobulin heavy constant µ; proto-oncogene diffuse B-cell lymphoma (DBL2); 26S protease regulatory subunit 4 (P26s4); serum albumin; haptoglobin; and two unknown proteins with isoelectronic point (pI) of 6.41 and molecular weight of 35.4 kDa, and pI of 8.05 and molecular weight of 27.4 kDa, respectively] were downregulated in MM compared with healthy controls. Expression of gel actin-related protein 2/3 complex subunit 1A (ARPC1A); immunoglobulin heavy constant γ 1; fibrinogen α chain (FGA) fragment D; and zinc finger protein 70 were increased in serum of MM patients. Protein expressions of ARPC1A, FGA, P26s4 and DBL2 were measured by immunoblotting in an independent cohort of 12 MM patients and 10 healthy controls. RT-qPCR analysis demonstrated that ARPC1A expression only mimicked protein expression, whereas FGA, PSMC1 (encoding P26s4) and MCF2 (encoding DBL2) did not exhibit significant changes in mRNA expression between control and MM samples. These proteins represent putative serological biomarkers for patients with MM.

The ubiquitin proteasome system (UPS) is a highly regulated mechanism of intracellular protein degradation and turnover. To evaluate the effect of crowding stress on the UPS in fish, grass carp (Ctenopharyngodon idellus) were randomly reared at low stocking density (LSD, 0.9 kg m−2) or high stocking density (HSD, 5.9 kg m−2) for 70 days. The expression of the genes regulating UPS, stress-related parameters, and profiles of amino acid in white muscle as well as growth rate of fish reared at two stocking densities were investigated. Fish exhibited significantly higher growth rate in the LSD group compared to the HSD group. Serum concentrations of cortisol, total protein, and glucose did not vary significantly in fish between two groups. There was no significant difference in the mRNA levels of nrf2, keap1, and hsp90 in white muscle of fish stocked at two densities at the endpoint of the experiment. In the UPS pathway, the expressions of ub, chip, psmc1 in the LSD group were significantly higher than those in the HSD group (P < 0.05). Ubiquitinated protein level and the content of 3-Methylhistidine elevated significantly in the LSD group (P < 0.05). The mRNA levels of mafbx, murf1, and s6k1 in the LSD group were significantly higher than those in HSD group (P < 0.05). These results illustrate that the fish cultured in lower stocking density would exhibit a greater growth rate and a fast protein turnover in muscle.

DNA ligase 4 deficiency (LIG4-SCID) causes lymphopenia (T-B-NK+) and a radiosensitive SCID (RS-SCID) phenotype. We demonstrate, for the first time, flow cytometric-based kinetic analysis of phosphorylated H2AX (γH2AX) in lymphocyte subsets, especially NK cells, for the assessment of LIG4-SCID. Measurement of phosphorylated (p) ATM, SMC1, and H2AX (γH2AX) was performed by flow cytometry to assess DNA repair defects in a 3-year-old girl. Functional assessment (phosphorylation) was measured in T and NK cells (B cells were absent) before irradiation (background control) or after low-dose (2Gy) irradiation (1 and 24 hours). We observed maximal γH2AX at 1 hour postirradiation, with dephosphorylation at 24 hours postirradiation in healthy control patients. The patient showed normal frequencies (percentage) of T cells and NK cells for γH2AX, but increased levels of γH2AX compared with control patients at 1 hour postirradiation. At 24 hours postirradiation, there was a lack of dephosphorylation in a substantial proportion of lymphocytes (with differences observed between T and NK cells) compared with healthy control patients. Although there was dephosphorylation of γH2AX at 24 hours in patient lymphocytes compared with 1 hour, the amount remained elevated at 24 hours compared with in control patients. The data from pATM and pSMC1 were uninformative. Flow-based kinetic analysis of γH2AX is a useful marker for the diagnosis of LIG4-SCID.

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1(G93A) cells. PC12-SOD1(G93A) cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1(G93A) cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS.

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30–150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox−)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox+)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox−)-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox−)-exo had enhanced proteolytic activity compared with HepAD38 (dox+)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox−)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox+)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox−)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.

Summary Due to genome instability, most cancers exhibit loss of regions containing tumor suppressor genes and collateral loss of other genes. To identify cancer-specific vulnerabilities that are the result of copy number losses, we performed integrated analyses of genome-wide copy number and RNAi profiles and identified 56 genes for which gene suppression specifically inhibited the proliferation of cells harboring partial copy number loss of that gene. These CYCLOPS ( c opy number alterations y ielding c ancer l iabilities o wing to p artial los s ) genes are enriched for spliceosome, proteasome, and ribosome components. One CYCLOPS gene, PSMC2 , encodes an essential member of the 19S proteasome. Normal cells express excess PSMC2, which resides in a complex with PSMC1, PSMD2, and PSMD5 and acts as a reservoir protecting cells from PSMC2 suppression. Cells harboring partial PSMC2 copy number loss lack this complex and die after PSMC2 suppression. These observations define a distinct class of cancer-specific liabilities resulting from genome instability.

The ubiquitin proteasome system (UPS) is a fundamental cellular pathway, degrading most unwanted intracellular soluble proteins. Dysfunction of the UPS has been associated with normal aging as well as various age-related pathological conditions, including chronic human neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to a significant interest in the involvement of this degradative system in neurones. We previously reported that the 26S proteasome was essential for neuronal homeostasis and survival in mouse brains following conditional genetic homozygous knockout of a key subunit of the multi-meric 26S proteasome (19S ATPase Psmc1). Here, we investigated the effects of Psmc1 heterozygosity in the mouse brain and primary mouse embryonic fibroblasts. Neuropathologically and biochemically, Psmc1 heterozygous (Psmc1(+/-)) knockout mice were indistinguishable from wild-type mice. However, we report a novel age-related accumulation of intraneuronal lysine 48-specific polyubiquitin-positive granular staining in both wild-type and heterozygous Psmc1 knockout mouse brain. In Psmc1(+/-) MEFs, we found a significant decrease in PSMC1 levels, altered 26S proteasome assembly and a notable G2/M cell cycle arrest that was not associated with an increase in the cell cycle regulatory protein p21. The disturbance in cell cycle progression may be responsible for the growth inhibitory effects in Psmc1(+/-) MEFs.

In the past twenty years, evidence has accumulated to show that ubiquitinated proteins are a consistent feature of the intraneuronal protein aggregates (inclusions) that characterize chronic neurodegenerative disease. These findings may indicate that age-related dysfunction of the 26S proteasome may be central to disease pathogenesis. The aggregate-prone proteins can also be eliminated by autophagy. We have used the Cre-recombinase/loxP genetic approach to ablate the proteasomal Psmc1 ATPase gene and deplete 26S proteasomes in neurons in different regions of the brain to mimic neurodegeneration. Deletion of the gene in dopaminergic neurons in the substantia nigra generates a new model of Parkinson disease. Ablation of the gene in the forebrain creates the first model of dementia with Lewy bodies. In both neuroanatomical regions, gene ablation causes the formation of Lewy-like inclusions together with extensive neurodegeneration. There is some evidence for neuronal autophagy in areas adjacent to inclusions. The models indicate that neuronal loss in neurodegenerative diseases can be attributed to proteasomal malfunction accompanied by Lewy-like inclusions as seen in dementia with Lewy bodies and Parkinson disease.

The 26S ATP-dependent protease is composed of a 20S catalytic proteasome and two PA700 regulatory modules; it plays a central role in many regulatory pathways, such as cell cycle regulation, differentiation, and apoptosis. The PA700 complex is composed of multiple subunits, including at least six related ATPases and approximately 15 non-ATPase polypeptides. PSMC1 (proteasome 26S subunit, ATPase, 1) is one of these ATPases. In this study, we amplified a fragment of 507 bp from intron 9 of the bovine PSMC1 gene and found a SNP (G/A) at position 216 in the PCR fragment. Genotyping of 138 animals from four beef breeds revealed that the average frequency for allele A (G-base) was 0.4271 (0.3269–0.5517); for allele B (A-base) it was 0.5729 (0.4483–0.6731). This SNP is significantly associated with average daily feed intake (P

// Shaogui Wang 1, 2 , Hong-Min Ni 2 , Kenneth Dorko 2 , Sean C. Kumer 3 , Timothy M. Schmitt 3 , Atta Nawabi 3 , Masaaki Komatsu 4 , Heqing Huang 1 , Wen-Xing Ding 2 1 Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China 2 Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA 3 Department of General Surgery, University of Kansas Medical Center, Kansas City, KS 66160, USA 4 Department of Biochemistry, School of Medicine Niigata University, Chuo-ku Niigata 951-8510, Japan Correspondence to: Heqing Huang, e-mail: huangheq@mail.sysu.edu.cn Wen-Xing Ding, e-mail: wxding@kumc.edu Keywords: alcohol, RIP3, steatosis, necroptosis, liver injury Received: July 28, 2015 Accepted: December 23, 2015 Published: January 12, 2016 ABSTRACT Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation.

In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.

Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be achieved. Here we developed and applied a dual-fluorescence-based Protein Turnover Assay (ProTA) to quantitatively profile global changes in human protein degradome upon BTZ-induced proteasomal inhibition. ProTA and subsequent network analyses delineate potential molecular basis for BTZ action and tumor drug resistance in BTZ chemotherapy. Finally, combined use of BTZ with drugs targeting the ProTA-identified key genes or pathways in BTZ action reduced BTZ resistance in multiple myeloma cells. Remarkably, BTZ stabilizes proteasome subunit PSMC1 and proteasome assembly factor PSMD10, suggesting a previously under-appreciated mechanism for regulating proteasome homeostasis. Therefore, ProTA is a novel tool for profiling human protein degradome to elucidate potential mechanisms of drug action and resistance, which might facilitate therapeutic development targeting proteostasis to treat human disorders.

Extracellular ATP has been implicated in a number of cellular events, including mammalian sperm function. The complement of ATP-dependent sperm proteins includes six subunits of the 26S proteasome, a multi-subunit protease specific to ubiquitinated substrate-proteins. Proteolysis of ubiquitinated proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis (AE) and sperm-zona pellucida (ZP) penetration. The 26S proteasome is uniquely present on the sperm acrosomal surface during mammalian, ascidian, and invertebrate fertilization. The proteasome is a multi-subunit protease complex of approximately 2 MDa composed of the 19S regulatory complex and a 20S proteolytic core. Integrity of the 19S complex is maintained by six 19S ATPase subunits (PSMC1 through PSMC6). Consequently, we hypothesized that fertilization will be blocked by the depletion of sperm-surface associated ATP (ssATP). Depletion of ssATP by the Solanum tuberosum apyrase, a 49 kDa, non-cell permeant enzyme, significantly reduced the ATP content measured by an adapted luminescence-ATP assay from which all permeabilizing agents were excluded. Addition of active apyrase to porcine in vitro fertilization (IVF) medium caused a concentration dependent reduction in the overall fertilization rate. No such outcomes were observed in control groups using heat-inactivated apyrase. Apyrase treatment altered the band pattern of 19S ATPase subunits PSMC1 (Rpt2) and PSMC4 (Rpt3) in Western blotting, suggesting that it had an effect on the integrity of the sperm proteasomal 19S complex. Apyrase only altered the proteasomal core activities slightly, since these activities are not directly dependent on external ATP. In contrast, sperm treatment with MG132, a specific inhibitor of the proteasomal core chymotrypsin-like activity, inhibited the target proteolytic activity, but also induced a compensatory elevation in proteasomal peptidyl-glutamyl peptide hydrolase activity. Altogether, the present data provide an important missing piece of evidence in support of the ssATP-dependent, proteasomal-proteolytic model of sperm-ZP interactions.

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation ofPsmc1(Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome andPsmc1conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and α-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Little is known about the factors that influence the proteasome structures in cells and their activity, although this could be highly relevant to cancer therapy. We have previously shown that, within minutes, irradiation inhibits substrate degradation by the 26S proteasome in most cell types. Here, we report an exception in U87 glioblastoma cells transduced to express the epidermal growth factor receptor vIII (EGFRvIII) mutant (U87EGFRvIII), which does not respond to irradiation with 26S proteasome inhibition. This was assessed using either a fluorogenic substrate or a reporter gene, the ornithine decarboxylase degron fused to ZsGreen (cODCZsGreen), which targets the protein to the 26S proteasome. To elucidate whether this was due to alterations in proteasome composition, we used quantitative reverse transcription-PCR to quantify the constitutive (X, Y, Z) and inducible 20S subunits (Lmp7, Lmp2, Mecl1), and 11S (PA28α and β) and 19S components (PSMC1 and PSMD4). U87 and U87EGFRvIII significantly differed in expression of proteasome subunits, and in particular immunosubunits. Interestingly, 2 Gy irradiation of U87 increased subunit expression levels by 16% to 324% at 6 hours, with a coincident 30% decrease in levels of the proteasome substrate c-myc, whereas they changed little in U87EGFRvIII. Responses similar to 2 Gy were seen in U87 treated with a proteasome inhibitor, NPI0052, suggesting that proteasome inhibition induced replacement of subunits independent of the means of inhibition. Our data clearly indicate that the composition and function of the 26S proteasome can be changed by expression of the EGFRvIII. How this relates to the increased radioresistance associated with this cell line remains to be established. (Mol Cancer Res 2008;6(3):426–34)

Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta-galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.

The hormone gastrin helps maintain gastric mucosal integrity by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and Regenerating protein 1 (Reg1) , but the mechanisms involved are incompletely understood. We hypothesized that the proteasome beta subunit PSMB1, recently implicated in the gastrin responsiveness of vesicular monoamine transporter 2, may be required for gastrin-mediated upregulation of PAI-2 and Reg1. PSMB1 was knocked down by siRNA in the gastric cancer cell line AGS-GR, which expresses the cholecystokinin 2 receptor, and the gastrin response to PAI-2 (1.6kb) and Reg1 (2.1kb) promoter-luciferase reporter constructs determined. In control cells (scrambled RNA) gastrin (2nM G17 for 6h) increased PAI-2 transcription by 9.2±1.5 fold (n=6), but in cells with PSMB1 knocked down (69.5±5.5% protein reduction), this was significantly reduced to 2.1±0.2 fold (p,0.001, ANOVA). In control cells, gastrin increased Reg1 transcription by 2.4±0.9 fold (n=9), but with PSMB1 knocked down, the response was abolished (1.2±0.1 fold, p,0.03). To investigate PSMB1 binding to the promoters, ChIP assays were performed using an anti-PSMB1 antibody for immunoprecipitation, or IgG control. In anti-PSMB1 precipitated chromatin from cells treated with gastrin (2nM G17 for 2h), there was a 7.9±2.5 (n=3) fold enrichment of DNA compared to control, determined by real time PCR using primers that generated a 250bp amplicon incorporating the region of the PAI-2 promoter known to respond to gastrin. Using primers generating a 218bp amplicon including the gastrin responsive region of the Reg1 promoter we observed 10.0±4.6 fold enrichment. Immunocytochemistry of unstimulated AGS-GR cells revealed distribution of PSMB1 throughout the cytoplasm and nucleus. After 2h stimulation with 2nM G17, cytoplasmic PSMB1 was barely detectable and nuclear staining was intensified; after 6h stimulation, PSMB1 was again visible in cytoplasm. Western blotting of nuclear and cytoplasmic extracts showed that the nuclear:cytoplasmic ratio of PSMB1 changed from 1.7±0.4 in untreated cells to 10.6±2.6 (n=7, p,0.01) after 2h stimulation with G17. This subcellular redistribution was not seen with alpha (PSMA5) or regulatory (PSMC1) proteasome subunits. PSMB1 distribution was unaffected by activation of other receptors expressed by AGS-GR cells, including histamine, epidermal growth factor, PGE2, or IL-8. The response to G17 was however mimicked by phorbol 12-myristate 13-acetate (0.1μM), and prevented by the protein kinase C (PKC) antagonist Ro-32-0432 (1μM). We conclude that proteasome beta subunits bind to the PAI-2 and Reg1 promoters in response to gastrin stimulation of AGSGR cells and are required for transcription. Gastrin induces a PKC-dependent subcellular redistribution of beta proteasome subunits that may be linked to their transcriptional functions.

The hormone gastrin helps maintain gastric mucosal integrity by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and Regenerating protein 1 (Reg1) , but the mechanisms involved are incompletely understood. We hypothesized that the proteasome beta subunit PSMB1, recently implicated in the gastrin responsiveness of vesicular monoamine transporter 2, may be required for gastrin-mediated upregulation of PAI-2 and Reg1. PSMB1 was knocked down by siRNA in the gastric cancer cell line AGS-GR, which expresses the cholecystokinin 2 receptor, and the gastrin response to PAI-2 (1.6kb) and Reg1 (2.1kb) promoter-luciferase reporter constructs determined. In control cells (scrambled RNA) gastrin (2nM G17 for 6h) increased PAI-2 transcription by 9.2±1.5 fold (n=6), but in cells with PSMB1 knocked down (69.5±5.5% protein reduction), this was significantly reduced to 2.1±0.2 fold (p,0.001, ANOVA). In control cells, gastrin increased Reg1 transcription by 2.4±0.9 fold (n=9), but with PSMB1 knocked down, the response was abolished (1.2±0.1 fold, p,0.03). To investigate PSMB1 binding to the promoters, ChIP assays were performed using an anti-PSMB1 antibody for immunoprecipitation, or IgG control. In anti-PSMB1 precipitated chromatin from cells treated with gastrin (2nM G17 for 2h), there was a 7.9±2.5 (n=3) fold enrichment of DNA compared to control, determined by real time PCR using primers that generated a 250bp amplicon incorporating the region of the PAI-2 promoter known to respond to gastrin. Using primers generating a 218bp amplicon including the gastrin responsive region of the Reg1 promoter we observed 10.0±4.6 fold enrichment. Immunocytochemistry of unstimulated AGS-GR cells revealed distribution of PSMB1 throughout the cytoplasm and nucleus. After 2h stimulation with 2nM G17, cytoplasmic PSMB1 was barely detectable and nuclear staining was intensified; after 6h stimulation, PSMB1 was again visible in cytoplasm. Western blotting of nuclear and cytoplasmic extracts showed that the nuclear:cytoplasmic ratio of PSMB1 changed from 1.7±0.4 in untreated cells to 10.6±2.6 (n=7, p,0.01) after 2h stimulation with G17. This subcellular redistribution was not seen with alpha (PSMA5) or regulatory (PSMC1) proteasome subunits. PSMB1 distribution was unaffected by activation of other receptors expressed by AGS-GR cells, including histamine, epidermal growth factor, PGE2, or IL-8. The response to G17 was however mimicked by phorbol 12-myristate 13-acetate (0.1μM), and prevented by the protein kinase C (PKC) antagonist Ro-32-0432 (1μM). We conclude that proteasome beta subunits bind to the PAI-2 and Reg1 promoters in response to gastrin stimulation of AGSGR cells and are required for transcription. Gastrin induces a PKC-dependent subcellular redistribution of beta proteasome subunits that may be linked to their transcriptional functions.

Mantle Cell Lymphoma (MCL) is a malignancy of mature B-cells. MCL has a poor prognosis and a limited response to traditional chemotherapy. Bortezomib (BZM), a new powerful inhibitor of the proteasome, can induce responses in up to 50% of relapsed MCL patients, suggesting that in at least half of the patients the lymphoma cells are intrinsically resistant to BZM or rapidly develop resistance during single agent therapy. To investigate possible mechanisms of BZM resistance, we cultured MCL cell lines continuously in sub-lethal concentrations of BZM that were then gradually increased. Resistance was slow to develop taking several months for truly resistant clones to grow out. We generated a bortezomib resistant (BR) clone of HBL-2 with an IC50 of 30nM compared to 5nM in the parental clone and several BR subclones of Jeko-1, the most resistant of which had an IC50 of 200nM compared to 3nM for the parental clone. All BR subclones also showed decreased sensitivity to three other proteasome inhibitors: MG-132, Lactacystin, and NLVS. The increase in IC50 to these drugs was between 3 and 8-fold, consistent with more off-target effects of these drugs compared to BZM. BAY11-7082, an inhibitor of NF-kB signaling, maintained its activity against the resistant cells. Resistance to BZM, once acquired, has remained stable over several months. This is remarkable because the resistant subclones grow significantly slower than the parental lines, even after having been removed from selection for extended periods of time. Consistent with slower cell proliferation, we found reduced Cyclin D1 protein expression in the BZM resistant Jeko clones; however, mRNA levels were comparable to the parental line, indicating that changes in Cyclin D1 protein translation and/or stability may be responsible for the decreased proliferation. BZM resistance has been associated with up-regulation of proteasome components and heat-shock proteins. Indeed, in the resistant HBL-2 subclone we found marked upregulation of two proteasome components (PSMA5 and PSMC1) and of Hsp70 by RT-PCR, but there was only a small change in Hsp70 protein expression. Nevertheless, upregulation of these genes could be part a more global gene expression response as seen with ER-stress and could thus reflect an adaptive change to BZM in the HBL-2 BR subclone. All three Jeko BR clones in contradistinction showed only minor changes in PSMA5, PSMC1 and Hsp70 mRNA expression and surprisingly had markedly reduced Hsp70 protein levels. Thus, in these subclones, BR resistance appears to correlate primarily with changes affecting cell cycle control. We conclude that resistance to BZM may be determined by several mechanisms that affect cell cycle control as well as expression of proteasome components and heat-shock proteins. While the slow development of resistance suggests adaptive changes, its persistence over time is more consistent with mutations or other genomic alterations that are not readily reversible. Ongoing studies aim to more precisely define the basis for BZM resistance in MCL.