Expression Of Proteasome Subunits Research Articles

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells.

Introduction: Proteasomes are multi-subunit protein complexes responsible for protein degradation in cells. Immunoproteasomes and intermediate proteasomes (together non-constitutive proteasomes) are specific forms of proteasomes frequently associated with immune response, antigen presentation, inflammation and stress. Expression of non-constitutive proteasome subunits has a prognostic value in several types of cancer. Thus, factors that modulate non-constitutive proteasome expression in tumors are of particular interest. Multikinase inhibitors (MKIs) demonstrate promising results in treatment of cancer. At the same time, their immunomodulatory properties and effects on non-constitutive proteasome expression in colorectal cancer cells are poorly investigated. Methods: Proteasome subunit expression in colorectal cancer was evaluated by bioinformatic analysis of available datasets. Two colorectal cancer cell lines, expressing fluorescent non-constitutive proteasomes were treated with multikinase inhibitors: regorafenib and sorafenib. The proteasome subunit expression was assessed by real-time PCR, Western blotting and flow cytometry. The proteasome activity was studied using proteasome activity-based probe and fluorescent substrates. Intracellular proteasome localization was revealed by confocal microscopy. Reactive oxygen species levels following treatment were determined in cells. Combined effect of proteasome inhibition and treatment with MKIs on viability of cells was estimated. Results: Expression of non-constitutive proteasomes is increased in BRAF-mutant colorectal tumors. Regorafenib and sorafenib stimulated the activity and synthesis of non-constitutive proteasomes in examined cell lines. MKIs induced oxidative stress and redistribution of proteasomes within cells. Sorafenib stimulated formation of cytoplasmic aggregates, containing proteolyticaly active non-constitutive proteasomes, while regorafenib had no such effect. MKIs caused no synergistic action when were combined with the proteasome inhibitor. Discussion: Obtained results indicate that MKIs might affect the crosstalk between cancer cells and immune cells via modulation of intracellular proteasome pool. Observed phenomenon should be considered when MKI-based therapy is applied.

Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes

BackgroundActivation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies.MethodsThis study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads.ResultsWe show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival.ConclusionsAltogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

Abstract 571: CRISPR/Cas9 screening identifies the proteasome subunit PSMC6 as key for the survival of cisplatin-resistant ovarian carcinoma cells

Abstract Ovarian carcinoma is a disease with poor prognosis, due to delays in diagnosis and the development of drug resistance. Since deregulation of the ubiquitin proteasome system may contribute to the development of drug resistance, the aim of this study was to investigate the interplay between expression of proteasome subunits and cisplatin resistance by utilizing a CRISPR/Cas9 dropout screen. Paired cisplatin-sensitive and -resistant cells (IGROV-1 and IGROV-1/Pt1) were transfected with Cas9 and transduced with gRNAs against proteasome components. Cells were collected at early (day 4) and late (day 21) time exposure and sequenced to identify genes necessary for survival. Various proteasome subunits were found to be essential for the survival of cisplatin-resistant ovarian carcinoma cells, including PSMA5, PSMC6, PSMD7, PSMD12 and PSMD14. Since PSMC6 knockout produced the greatest growth inhibitory effects, it was selected for functional studies utilizing conventional RNA interference. When IGROV-1 and IGROV-1/Pt1 cells were transfected with small interfering RNAs (siRNAs) targeting PSMC6, efficient down-regulation on the mRNA and protein level was observed. Phenotypic analyses of PSMC6 knockdown cells indicated reduced clonogenicity using both 2D and soft agar assays in parental and resistant cells associated with down-regulation of the canonical MAPK pathway. A slight but not significant increase in sensitivity to cisplatin was observed upon PSMC6 silencing in resistant cells using clonogenic assays. Finally, we carried out a pilot study using ovarian carcinoma specimens (n = 134) obtained through the institutional biobank. The samples were representative of the frequency occurrence of the various histological ovarian carcinoma subtypes. We found that PSMC6 expression, evaluated by qRT-PCR, was associated with tumor stage. These findings suggest that PSMC6 is linked to ovarian carcinoma aggressiveness and that PSMC6 targeting may be an interesting therapeutic strategy. Citation Format: Matteo Costantino, Padraig D'arcy, Luca Mirra, Cristina Corno, Giovanni L. Beretta, Johannes Gubat, Pietro Pratesi, Chiara M. Ciniselli, Paolo Verderio, Stig Linder, Paola Perego. CRISPR/Cas9 screening identifies the proteasome subunit PSMC6 as key for the survival of cisplatin-resistant ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 571.

Abstract 7271: Proteasome inhibitor FHND6091: A potent therapeutic candidate forPTEN-deficient cholangiocarcinoma

Abstract Background: Cholangiocarcinoma (CCA), characterized by high genetic heterogeneity, poses a significant challenge due to limited therapeutic options. Notably, the tumor suppressor gene PTEN is frequently altered in more than 50% of CCA clinical specimens. PTEN deficiency enhances proteasome subunit expression and proteasome proteolytic activity, and facilitates the potential therapeutic efficacy of proteasome inhibitors in CCA. The proteasome inhibitor FHND6091, a novel drug developed by Jiangsu Chia Tai Fenghai Pharmaceutical Co., Ltd., has obtained IND approval from NMPA and is currently undergoing a first-in-human phase 1 trial for multiple myeloma (MM) and mantle cell lymphoma (MCL). Method: This study employed a comprehensive approach, encompassing both in vitro and in vivo pharmacology studies. The investigation involved purified human proteasome activity assays, cell viability assays, and in vivo anti-tumor activity studies utilizing a cholangiocarcinoma patient-derived xenograft (CCA PDX) model with PTEN deficiency. Results: FHND6091 exhibited potent inhibitory effects on the β5 subunit of the 20S proteasome, with IC50 value of 2.22 nM. In cell viability assessments, FHND6091 demonstrated selective efficacy in PTEN-deficient CCA cell lines, with IC50 value of 5.87 nM in HCCC-9810 (PTEN deletion, homozygous loss of exon 6 to 9) and 10.08 nM in RBE (PTEN intact expression). In a PDX xenograft model with PTEN deficiency, gavage administration of FHND6091 BIW at 1.0 mg/kg (first week)/1.5 mg/kg (subsequent two weeks) exhibited advanced anti-tumor activity (TGI = 52.32%) compared to intraperitoneal administration of Bortezomib at 0.5 mg/kg BIW (TGI = 31.88%). Tissue distribution studies in rats revealed the preference of FHND6091 for liver accumulation, with an exposure (AUC0-t) approximately 44 times that of plasma after a single dose. These data corroborated that FHND6091 could achieve a specific level of exposure in the target organ liver. Conclusion: FHND6091, characterized by its irreversibility, high potency and oral bioavailability, emerges as a promising proteasome inhibitor for the treatment of both hematologic and solid tumors. These findings strongly support FHND6091 as a potential candidate for the treatment of CCA in clinical settings. Furthermore, the regulating of sensitivity to proteasome inhibitors by the PTEN status extends beyond cholangiocarcinoma to encompass various human cancers, such as ovarian, prostate, and digestive system malignancies. This broader implication establishes a theoretical foundation supporting the potential efficacy of FHND6091 as a therapeutic agent for solid tumors. Citation Format: Yongqiang Zhu, Jingmiao Shi, Chenhui Li. Proteasome inhibitor FHND6091: A potent therapeutic candidate forPTEN-deficient cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7271.

Shikonin Induces Glioma Necroptosis, Stemness Decline, and Impedes (Immuno)Proteasome Activity

Gliomas, the most prevalent primary intracranial tumors, exhibit notable features such as heightened malignancy, rapid recurrence, and elevated mortality rates. Presently, standard therapeutic approaches yield limited curative outcomes. Shikonin, an extract derived from traditional Chinese medicine, demonstrates notable bioactivity against various tumors, including gliomas. This study elucidates Shikonin’s capacity to effectively induce necroptosis in glioma cells, concurrently mitigating glioma stemness, as evidenced by diminished levels of stem cell markers, namely SOX2, CD44, CHI3L1, and CD24. Our findings indicate that Shikonin-induced programed necrosis leads to a downregulation of proteasome activity and a decrease in the expression of immune proteasome subunits PSMB8/9/10 and PSME1/2/3, contributing to the attenuation of stemness in gliomas. This study comprehensively investigates the interplay between (immuno)proteasome dynamics, Shikonin-mediated necroptosis, and the consequential reduction in glioma stemness, both in vitro and in vivo. The discussion extends to the potential of Shikonin as a promising therapeutic agent in the management of gliomas, offering a novel avenue for drug development in this challenging clinical context.

Combined Genetic Association and Differed Expression Analysis of UBE2L3 Uncovers a Genetic Regulatory Role of (Immuno)proteasome in IgA Nephropathy

Introduction: IgA nephropathy (IgAN) is a leading cause of end-stage renal disease. The exact pathogenesis of IgAN is not well defined, but some genetic studies have led to a novel discovery that the (immuno)proteasome probably plays an important role in IgAN. Methods: We firstly analyzed the association of variants in the UBE2L3 region with susceptibility to IgAN in 3,495 patients and 9,101 controls, and then analyzed the association between lead variant and clinical phenotypes in 1,803 patients with regular follow-up data. The blood mRNA levels of members of the ubiquitin-proteasome system including UBE2L3 were analyzed in peripheral blood mononuclear cells from 53 patients and 28 healthy controls. The associations between UBE2L3 and the expression levels of genes involved in Gd-IgA1 production were also explored. Results: The rs131654 showed the most significant association signal in UBE2L3 region (OR: 1.10, 95% CI: 1.04–1.16, p = 2.29 × 10−3), whose genotypes were also associated with the levels of Gd-IgA1 (p = 0.04). The rs131654 was observed to exert cis-eQTL effects on UBE2L3 in various tissues and cell types, particularly in immune cell types in multiple databases. The UBE2L3, LUBAC, and proteasome subunits were highly expressed in patients compared with healthy controls. High expression levels of UBE2L3 were not only associated with higher proteinuria (r = 0.34, p = 0.01) and lower eGFR (r = −0.28, p = 0.04), but also positively correlated with the gene expression of LUBAC and other proteasome subunits. Additionally, mRNA expression levels of UBE2L3 were also positively correlated with IL-6 and RELA, but negatively correlated with the expression levels of the key enzyme in the process of glycosylation including C1GALT1 and C1GALT1C1. Conclusion: In conclusion, by combined genetic association and differed expression analysis of UBE2L3, our data support a role of genetically conferred dysregulation of the (immuno)proteasome in regulating galactose-deficient IgA1 in the development of IgAN.

Knockout of PA200 improves proteasomal degradation and myelination in a proteotoxic neuropathy.

The cellular response to a decrease in protein degradation by 26S proteasomes in chronic diseases is poorly understood. Pharmacological inhibition of proteasomes increases the expression of proteasome subunits and Proteasome Activator 200 (PA200), an alternative proteasome activator. In the S63del mouse model of the peripheral neuropathy Charcot Marie Tooth 1B (CMT1B), proteasomal protein degradation is decreased and proteasome gene expression is increased. Here, we show an increase in PA200 and PA200-bound proteasomes in the peripheral nerves of S63del mice. To test genetically whether the upregulation of PA200 was compensatory, we generated S63del//PA200-/- mice. Unexpectedly, in the sciatic nerves of these mice, there was greater proteasomal protein degradation than in S63del, less polyubiquitinated proteins and markers of the unfolded protein response, and a greater amount of assembled, active 26S proteasomes. These changes were not seen in PA200-/- controls and were therefore specific to the neuropathy. Furthermore, in S63del//PA200-/- mice, myelin thickness and nerve conduction were restored to WT levels. Thus, the upregulation of PA200 is maladaptive in S63del mice and its genetic ablation prevented neuropathy.

MiR-383 sensitizes osteosarcoma cells to bortezomib treatment via down-regulating PSMB5.

Proteasome inhibition is a promising strategy for cancer therapy. Bortezomib, which primarily targets the chymotrypsin-like activity of PSMB5, has demonstrated efficacy in various tumors. However, there is variable sensitivity to bortezomib, which could be attributed, in part, to variations in the expression of proteasome subunits. In this study, we investigated whether miR-383 affects the expression of proteasome subunits in osteosarcoma (OS) cells, and if so, whether OS cells display differential sensitivity to bortezomib concerning miR-383 expression. We detected a decreased miR-383 expression in OS cells and tissues. Then we found a negative correlation between the cytotoxicity of bortezomib and the expression level of the proteasome 20S core particle subunit β5 (PSMB5). Intriguingly, we identified PSMB5 as a direct target of miR-383. Increased expression of miR-383 resulted in decreased PSMB5 expression and increased sensitivity to bortezomib in OS cells. In summary, our findings present the initial comprehensive analysis of the function of miR-383 in OS. The outcomes indicate that miR-383 may augment the anticancer effect of bortezomib through PSMB5 repression, offering a novel therapeutic approach in OS and a fresh pathway for proteasome regulation.

A novel autism-associated UBLCP1 mutation impacts proteasome regulation/activity

The landscape of autism spectrum disorder (ASD) in Lebanon is unique because of high rates of consanguinity, shared ancestry, and increased remote consanguinity. ASD prevalence in Lebanon is 1 in 68 with a male-to-female ratio of 2:1. This study aims to investigate the impact of an inherited deletion in UBLCP1 (Ubiquitin-Like Domain-Containing CTD Phosphatase 1) on the ubiquitin-proteasome system (UPS) and proteolysis. Whole exome sequencing in a Lebanese family with ASD without pathogenic copy number variations (CNVs) uncovered a deletion in UBLCP1. Functional evaluation of the identified variant is described in fibroblasts from the affected. The deletion in UBLCP1 exon 10 (g.158,710,261CAAAG > C) generates a premature stop codon interrupting the phosphatase domain and is predicted as pathogenic. It is absent from databases of normal variation worldwide and in Lebanon. Wild-type UBLCP1 is widely expressed in mouse brains. The mutation results in decreased UBLCP1 protein expression in patient-derived fibroblasts from the autistic patient compared to controls. The truncated UBLCP1 protein results in increased proteasome activity decreased ubiquitinated protein levels, and downregulation in expression of other proteasome subunits in samples from the affected compared to controls. Inhibition of the proteasome by using MG132 in proband cells reverses alterations in gene expression due to the restoration of protein levels of the common transcription factor, NRF1. Finally, treatment with gentamicin, which promotes premature termination codon read-through, restores UBLCP1 expression and function. Discovery of an ASD-linked mutation in UBLCP1 leading to overactivation of cell proteolysis is reported. This, in turn, leads to dysregulation of proteasome subunit transcript levels as a compensatory response.

IMPACT OF MITOCHONDRIAL MALFUNCTION IN AGING IS MEDIATED BY IMPAIRED LYSOSOMES AND ENDOPLASMIC RETICULUM

Abstract The hallmarks of aging are well established, and include mitochondrial malfunction, deregulated nutrient sensing, loss of proteostasis, and senescence, among others. While these hallmarks are all part of the aging process, they have typically been studied independently. We have found that mitochondrial malfunction can affect lysosomal activity, impacting nutrient sensing, and our that the crosstalk between mitochondria and other organelles such as endoplasmic reticulum and lysosomes affects cellular senescence, proteostasis and intercellular communication. Chronic mitochondrial malfunction causes perturbations in the lysosomal membrane which affect the function of this organelle as well as the recruitment of folliculin (FLCN) to the lysosomes. FLCN regulates the balance between anabolism (stimulating mTORC1) and catabolism (repressing AMPK). Increased recruitment of FLCN to the lysosomal membrane in cells subjected to chronic mitochondrial malfunction results in hyperactive mTORC1 and inhibition of AMPK signaling, thus aberrantly promoting anabolism under conditions that are not optimal. Furthermore, peroxisomal β-oxidation increases, and expression of proteasome subunits decreases, with consequent perturbation of proteostasis. Overall, the impact of chronic mitochondrial malfunction over lysosomes triggers multiple perturbations in organelles and organelle crosstalk that culminate in aberrant nutrient sensing, metabolic rewiring, proteostasis impairment, senescence and release of sterile inflammation-related proteins to the extracellular medium that denote alterations in intercellular communication. We will build on these findings to explore an overall picture of organelle homeostasis in aging and in age-related diseases.

A Novel Indirubin-3-Monoxime Derivative Functions As Regulator of Proteostasis Via Targeting TRIM28 in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains an incurable plasma cell malignancy. Agents disturbing the proteostasis play indispensable roles in MM treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) works as a novel proteasome inhibitor that effectively inhibits MM cell growth. In this study, we aim to investigate the improvement of I3MO combined with histone deacetylase 6 (HDAC6) inhibitors, and clarified the molecular mechanisms. Methods: We constructed a compound 8b combined with I3MO and HADC6 inhibitor. The anti-MM effects of compound 8b were investigated in vivo and in vitro studies. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. RNA-seq and Dual-luciferase reporter gene assay were performed to identify the downstream targets of 8b treatment. We constructed MM cell lines with TRIM28 overexpression or shRNA knockdown. We further explored the function of TRIM28, one of the targets of 8b treatment, by ChIP-seq and IP-MS. Results: The novel I3MO derivative 8b more efficiently suppressed myeloma cell growth in vitro and in vivo. Compound 8b not decreased proteasome activity but inhibited the formation of aggresomes and autophagosomes. Interesting, we found that compound 8b efficiently inhibited TRIM28 promoter activity and repressed TRIM28 transcription level further. GEO data analysis demonstrated a positive correlation between TRIM28 and the expression of proteasome subunits as well as autophagy-related genes in MM. Further, we utilized ChIP-seq to confirm that TRIM28 could bind to the promoter region of multiple proteasome subunits-encoding genes, including PSMB1. Our data indicated that TRIM28 knockdown not only decreased the proteasome activity, but inhibited the formation of autophagosomes. Knockdown TRIM28 induced a higher sensitivity to BTZ treatment in MM cells. Furthermore, IP-MS with Co-IP analysis showed that TRIM28 interacts with 14-3-3ζ. TRIM28 acts as an E3 ligase in mediating the ubiquitination and degradation of 14-3-3ζ, a well-known negative regulator of autophagy. 14-3-3ζ degradation induced by TRIM28 activates autophagy in MM cells. These data suggested that TRIM28 suppressed by compound 8b would involve in proteostasis by regulation of proteasome activity and autophagy. Conclusions: Our study identified a novel compound 8b, I3MO combined with HDAC6i, efficiently suppressed MM cells survival. Additionally, we for the first time provided novel important insights for the roles of TRIM28 in MM pathobiology by regulation of proteasome activity and autophagy.

Role of NFE2L1 in the Regulation of Proteostasis: Implications for Aging and Neurodegenerative Diseases.

A hallmark of aging and neurodegenerative diseases is a disruption of proteome homeostasis ("proteostasis") that is caused to a considerable extent by a decrease in the efficiency of protein degradation systems. The ubiquitin proteasome system (UPS) is the major cellular pathway involved in the clearance of small, short-lived proteins, including amyloidogenic proteins that form aggregates in neurodegenerative diseases. Age-dependent decreases in proteasome subunit expression coupled with the inhibition of proteasome function by aggregated UPS substrates result in a feedforward loop that accelerates disease progression. Nuclear factor erythroid 2- like 1 (NFE2L1) is a transcription factor primarily responsible for the proteasome inhibitor-induced "bounce-back effect" regulating the expression of proteasome subunits. NFE2L1 is localized to the endoplasmic reticulum (ER), where it is rapidly degraded under basal conditions by the ER-associated degradation (ERAD) pathway. Under conditions leading to proteasome impairment, NFE2L1 is cleaved and transported to the nucleus, where it binds to antioxidant response elements (AREs) in the promoter region of proteasome subunit genes, thereby stimulating their transcription. In this review, we summarize the role of UPS impairment in aging and neurodegenerative disease etiology and consider the potential benefit of enhancing NFE2L1 function as a strategy to upregulate proteasome function and alleviate pathology in neurodegenerative diseases.

Interplay between proteasome function and inflammatory responses in e-cig vapor condensate-challenged lung epithelial cells.

Exposure to cigarettes and other nicotine-based products results in persistent inflammation in the lung. In recent years, electronic cigarettes (e-cigs) have become extremely popular among adults and youth alike. E-cigarette vapor-induced oxidative stress promotes protein breakdown, DNA damage and cell death, culminating in a variety of respiratory diseases. The proteasome, a multi-catalytic protease, superintends protein degradation within the cell. When cells are stimulated with inflammatory cytokines such as IFN-γ and TNF-α, the constitutive catalytic proteasome subunits are replaced by the inducible subunits-low-molecular mass polypeptide (LMP)2 (β1i), multi-catalytic endopeptidase complex-like (MECL)1 (β2i), and LMP7 (β5i), which are required for the production of certain MHC class I-restricted T-cell epitopes. In this study, we used human alveolar epithelial cells (A549) and exposed them to filtered air or (1%) tobacco-flavored (TF) electronic cigarette vapor condensate(ECVC) ± nicotine (6mg/ml) (TF-ECVC ± N) for 24h. We observed an increase in the levels of IFN-γ, TNF-α, and inducible proteasome subunits (LMP7/PSMB8, LMP2/PSMB9, MECL1/PSMB10), and a reduced expression of constitutive proteasome subunits (β1/PSMB6 and β2/PSMB7) in challenged A549 cells. Interestingly, knockdown of the inducible proteasome subunit LMP7 reversed ECVC-induced expression of NADPH oxidase and immunoproteasome subunits in A549 cells. In addition, pre-exposure to an LMP7 inhibitor (ONX-0914) abrogated the mRNA expression of several NOX subunits and rescued the excessive production/release of inflammatory cytokines/chemokines (IL-6, IL-8, CCL2, and CCL5) in ECVC-challenged cells. Our findings suggest an important role of LMP7 in regulating the expression of inflammatory mediators during ECVC exposure. Overall, our results provide evidence for proteasome-dependent ROS-mediated inflammation in ECVC-challenged cells.

UBE4A catalyzes NRF1 ubiquitination and facilitates DDI2-mediated NRF1 cleavage

The transcription factor nuclear factor erythroid 2 like 1 (NFE2L1 or NRF1) regulates constitutive and inducible expression of proteasome subunits and assembly chaperones. The precursor of NRF1 is integrated into the endoplasmic reticulum (ER) and can be retrotranslocated from the ER to the cytosol where it is processed by ubiquitin-directed endoprotease DDI2. DDI2 cleaves and activates NRF1 only when NRF1 is highly polyubiquitinated. It remains unclear how retrotranslocated NRF1 is primed with large amount of ubiquitin and/or very long polyubiquitin chain for subsequent processing. Here, we report that E3 ligase UBE4A catalyzes ubiquitination of retrotranslocated NRF1 and promotes its cleavage. Depletion of UBE4A reduces the amount of ubiquitin modified on NRF1, shortens the average length of polyubiquitin chain, decreases NRF1 cleavage efficiency and causes accumulation of non-cleaved, inactivated NRF1. Expression of a UBE4A mutant lacking ligase activity impairs the cleavage, likely due to a dominant negative effect. UBE4A interacts with NRF1 and the recombinant UBE4A can promote ubiquitination of retrotranslocated NRF1 in vitro. In addition, knocking out UBE4A reduces transcription of proteasomal subunits in cells. Our results indicate that UBE4A primes NRF1 for DDI2-mediated activation to facilitate expression of proteasomal genes.

The role of proteasome in muscle wasting of experimental arthritis

BackgroundRheumatoid arthritis is an autoimmune inflammatory disease that often leads patients to muscle impairment and physical disability. This study aimed to evaluate changes in the activity of proteasome system in skeletal muscles of mice with collagen-induced arthritis (CIA) and treated with etanercept or methotrexate.MethodsMale DBA1/J mice were divided into four groups (n = 8 each): CIA-Vehicle (treated with saline), CIA-ETN (treated with etanercept, 5.5 mg/kg), CIA-MTX (treated with methotrexate, 35 mg/kg) and CO (healthy control group). Mice were treated two times a week for 6 weeks. Clinical score and hind paw edema were measured. Muscles were weighted after euthanasia and used to quantify proteasome activity, gene (MuRF-1, PMSα4, PSMβ5, PMSβ6, PSMβ7, PSMβ8, PSMβ9, and PSMβ10), and protein (PSMβ1, PSMβ5, PSMβ1i, PSMβ5i) expression of proteasome subunits.ResultsBoth treatments slowed disease development, but only CIA-ETN maintained muscle weight compared to CIA-MTX and CIA-Vehicle groups. Etanercept treatment showed caspase-like activity of 26S proteasome similar to CO group, while CIA-Vehicle and CIA-MTX had higher activity compared to CO group (p: 0.0057). MuRF-1 mRNA expression was decreased after etanercept administration compared to CIA-Vehicle and CO groups (p: 0.002, p: 0.007, respectively). PSMβ8 and PSMβ9 mRNA levels were increased in CIA-Vehicle and CIA-MTX compared to CO group, while CIA-ETN presented no difference from CO. PMSβ6 mRNA expression was higher in CIA-Vehicle and CIA-MTX groups than in CO group. Protein levels of the PSMβ5 subunit were increased in CO group compared to CIA-Vehicle; after both etanercept and methotrexate treatments, PSMβ5 expression was higher than in CIA-Vehicle group and did not differ from CO group expression (p: 0.0025, p: 0.001, respectively). The inflammation-induced subunit β1 (LMP2) was enhanced after methotrexate treatment compared to CO group (p: 0.043).ConclusionsThe results of CIA-Vehicle show that arthritis increases muscle proteasome activation by enhanced caspase-like activity of 26S proteasome and increased PSMβ8 and PSMβ9 mRNA levels. Etanercept treatment was able to maintain the muscle weight and to modulate proteasome so that its activity and gene expression were compared to CO after TNF inhibition. The protein expression of inflammation-induced proteasome subunit was increased in muscle of CIA-MTX group but not following etanercept treatment. Thus, anti-TNF treatment may be an interesting approach to attenuate the arthritis-related muscle wasting.

The Proteasome and Ageing.

The proteasome is a multi-subunit proteolytic complex that functions to degrade normal proteins for physiological regulation and to eliminate abnormal proteins for cellular protection. Generally, the proteasome targets substrate proteins that are marked by attachment of multiple ubiquitin molecules. In various types of cells in an organism, damage to proteins occurs both from internal sources such as reactive oxygen species and from external ones such as UV radiation from the sun. The proteasome functions to protect the cells by degrading damaged proteins. With ageing, however, the capacity of the proteasome to degrade damaged proteins is reduced as indicated by evidence gathered by many studies. Studies on ageing in muscle, skin, and brain show that with age catalytic activity of the proteasome is decreased and the expression of proteasome subunits is altered. Age-related accumulation of damaged or misfolded proteins causes further reduction of proteasome activity. Abnormal proteins also accumulate as a result of age-related neurodegenerative diseases. Deficits in proteasome activity might be responsible for accumulation of protein aggregates and thus contribute to the pathology. Results from several studies suggest a link between the proteasome and longevity. This chapter reviews the various ways in which the proteasome is associated with the ageing process and examines evidence gathered from investigations on cultured cells, model organisms, and humans.

Proteasome Dysfunction Leads to Suppression of the Hypoxic Response Pathway in Arabidopsis

Proteasome is a large proteolytic complex that consists of a 20S core particle (20SP) and 19S regulatory particle (19SP) in eukaryotes. The proteasome degrades most cellular proteins, thereby controlling many key processes, including gene expression and protein quality control. Proteasome dysfunction in plants leads to abnormal development and reduced adaptability to environmental stresses. Previous studies have shown that proteasome dysfunction upregulates the gene expression of proteasome subunits, which is known as the proteasome bounce-back response. However, the proteasome bounce-back response cannot explain the damaging effect of proteasome dysfunction on plant growth and stress adaptation. To address this question, we focused on downregulated genes caused by proteasome dysfunction. We first confirmed that the 20SP subunit PBE is an essential proteasome subunit in Arabidopsis and that PBE1 mutation impaired the function of the proteasome. Transcriptome analyses showed that hypoxia-responsive genes were greatly enriched in the downregulated genes in pbe1 mutants. Furthermore, we found that the pbe1 mutant is hypersensitive to waterlogging stress, a typical hypoxic condition, and hypoxia-related developments are impaired in the pbe1 mutant. Meanwhile, the 19SP subunit rpn1a mutant seedlings are also hypersensitive to waterlogging stress. In summary, our results suggested that proteasome dysfunction downregulated the hypoxia-responsive pathway and impaired plant growth and adaptability to hypoxia stress.

19S Proteasome Subunits as Oncogenes and Prognostic Biomarkers in FLT3-Mutated Acute Myeloid Leukemia (AML)

26S proteasome non-ATPase subunits 1 (PSMD1) and 3 (PSMD3) were recently identified as prognostic biomarkers and potential therapeutic targets in chronic myeloid leukemia (CML) and multiple solid tumors. In the present study, we analyzed the expression of 19S proteasome subunits in acute myeloid leukemia (AML) patients with mutations in the FMS-like tyrosine kinase 3 (FLT3) gene and assessed their impact on overall survival (OS). High levels of PSMD3 but not PSMD1 expression correlated with a worse OS in FLT3-mutated AML. Consistent with an oncogenic role for PSMD3 in AML, shRNA-mediated PSMD3 knockdown impaired colony formation of FLT3+ AML cell lines, which correlated with increased OS in xenograft models. While PSMD3 regulated nuclear factor-kappa B (NF-κB) transcriptional activity in CML, we did not observe similar effects in FLT3+ AML cells. Rather, proteomics analyses suggested a role for PSMD3 in neutrophil degranulation and energy metabolism. Finally, we identified additional PSMD subunits that are upregulated in AML patients with mutated versus wild-type FLT3, which correlated with worse outcomes. These findings suggest that different components of the 19S regulatory complex of the 26S proteasome can have indications for OS and may serve as prognostic biomarkers in AML and other types of cancers.

High Immunoproteasome Expression As Indicator for Sensitivity to Bortezomib-Containing Chemotherapy in Newly Diagnosed, Standard and Intermediate Risk, T-Cell Acute Lymphoblastic Leukemia Patients from Children's Oncology Group Trial AALL1231

Background: Proteasome inhibition by bortezomib (BTZ) has previously been shown to be beneficial for a subset of patients with relapsed/refractory acute leukemia. Several (pre)clinical studies showed that relatively high immunoproteasome expression (β1i and β5i) may serve as a biomarker for BTZ-sensitivity and aid to select patients that may respond well to BTZ-containing therapy (Cloos J et al. Cancer Metastasis Rev. 2017). In this study we investigated whether we could confirm these findings in newly diagnosed T-cell acute lymphoblastic leukemia patients (T-ALL) enrolled in the COG-AALL1231 phase 3 clinical trial (Teachey DT et al. J Clin Oncol 2022). In that trial, patients in treatment arm A received augmented Berlin-Frankfurt-Münster (aBFM) therapy, while for those in treatment arm B, BTZ was added to the aBFM regimen. Methods: Baseline immuno- and constitutive proteasome subunit expression was determined in bone marrow samples of 77 standard and intermediate risk T-ALL patients using Western blot. To correct for potential variation between blots, two cell lines were included as internal quantification controls, i.e. the human (BTZ-sensitive) T-ALL cell line CCRF-CEM (CEM-WT) and its subline (CEM-BTZ200) with acquired resistance to 200 nM BTZ. Protein bands were quantified and calculated relative to β-actin and the subunit expression of CEM-WT. Mann-Whitney U and log-rank tests were used for between group comparisons of proteasome subunit expressions and event free survival (EFS), respectively. Induction failure, relapse and death (induction and remission) were considered as EFS events. Results: In the bone marrow samples, a very strong correlation was observed between immunoproteasome β5i and β1i subunit expression (R=0.9, P<0.0001, Figure 1A). The correlation for constitutive subunits was R=0.7, P<0.0001. Interestingly, females had significantly higher β1i and β5i immunoproteasome levels compared to males with medians of 5.1 (range: 0.9-18.7) and 1.6 (0.3-17.9) respectively for the β1i subunit (P =0.05), and medians of 4.8 (1.9-16.1) and 1.8 (0.6-10.8) respectively for the β5i subunit (P =0.0084). Three year EFS was used as a measure for response to therapy. In patients treated with the BTZ-containing therapy (arm B), higher immunoproteasome expression levels (>0.8, based on Max-stat analysis) correlated with an improved EFS (P=0.034 for β1i and P=0.011 for β5i, Figure 1B). In contrast, in treatment arm A, higher immunoproteasome expression levels were associated with a relatively poor EFS, although this difference was not statistically significant likely due to low patient numbers in the low immunoproteasome subunit expression group in arm A (β1i N=5 and β5i N=1, for arm B β1i N=6 and β5i N=4). No other factors, including age, ethnicity, and gender, were found to be significantly associated with EFS. Discussion: These results support our hypothesis that patients with higher immunoproteasome expression levels are responsive to proteasome inhibitors such as BTZ, with better EFS after BTZ-containing therapy. When comparing the patients with high immunoproteasome expression of arm A versus arm B, the latter displays a better EFS (3 years EFS 96% for arm B vs 80% for arm A, P=0.08). Although not statistically significant, this factor may be indicative of a beneficial effect of BTZ-treatment in this subgroup of patients. The limitation of this study is the relatively low number of samples in each subgroup, in particular the patients with low expression of immunoproteasome subunits. Since most patients show a high level of immunoproteasome, only the few with low expression might not benefit from BTZ treatment and may be allocated for alternative strategies. The relevance of the immunoproteasome for BTZ sensitivity in these samples will be further examined by proteasome subunit catalytic activity measurements with subunit-specific substrates and other known factors contribution to diminished BTZ sensitivity (Niewerth et al. Drug Resist Updates 2015). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of the Proteasome Subunits PSMB4 and PSMD4 As Novel Targets in Multiple Myeloma Patients Carrying 1q21 Amplification

Gain and/or amplification of the 1q21 (1q21+) region is one of the most frequent secondary cytogenetic abnormalities present in multiple myeloma (MM) patients. Interestingly, it has been reported that the incidence of the amplification of chromosome 1q (Amp1q) in patients with pre-malignant smoldering myeloma (SMM) is about 40%. Several data indicate that the presence of Amp1q is associated with a worse clinical outcome and a poor response to therapies including proteasome inhibitors (PIs). Recently, a study showed that newly diagnosed MM (NDMM) carrying Amp1q receiving carfilzomib-based treatment have an early disease progression and shorter overall survival, compared to those with 1q21 gain. The genes driving amplification of the 1q21 region have not been fully elucidated. This study aims to identify novel targets in the 1q21 region possibly correlated to PIs treatment. We purified CD138+ plasma cells (PCs) by immunomagnetic beads from bone marrow (BM) aspirates. We used a total cohort of 29 MM patients: 11 SMM and 18 NDMM. Fluorescent in situ hybridization analysis was performed on all the patients. 48% (14/29) patients carried 1q21+. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The smar package was used to identify differentially expressed genes between 1q21+ and control samples. The expression profile of our cohort showed that the expression of both proteasome subunits, PSMB4 and PSMD4 was significantly upregulated in MM patients Amp1q compared to control patients (PSMB4 p=0.0006; PSMD4p=<0.0001 respectively). Additionally, we found a strong positive correlation between gene expression levels and 1q21 copy number for the proteasome subunits PSMB4 (p=<0.0001, r=0.5631) and PSMD4 (p=<0.0001, r=0.6391). Interestingly, the PSMB4 and PSMD4 expression levels were independent of the disease stage (SMM vs MM) and the expression was driven exclusively by the 1q21 copy number. We evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 wild-type MM cell line (OCI-MY5) and a panel of MM cell lines carrying 1q21+. Our results showed that PSMB4 and PSMD4 expression levels were higher in cell lines with Amp1q, following a 1q21 copy number fashion. Subsequently, we assessed the role of PSMB4 and PSMD4 to the sensibility of PI bortezomib in two myeloma cell lines carrying 1q21+. Our results showed that bortezomib treatment downregulates the protein expression of PSMD4 while PSMB4 was unaffected. Moreover, the MTT viability test correlated with the anti-myeloma effect of bortezomib. Furthermore, we generated a JJN3 PSMB4 and PSMD4-knockdown MM cell line using short hairpin RNA (shRNA) lentivectors. An empty vector was used as a control plasmid. Cell viability and apoptosis assays were performed using MTT assay and Apo 2.7 staining. Our functional studies showed that shPSMB4 and shPSMD4 decreased MM cell viability when treated with PI. Moreover, both shPSMB4 and shPSMD4 knockdown cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation. In conclusion, our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in MM patients carrying 1q21+, correlated with 1q21 copy number but not with disease stage. Therefore, targeting PSMB4 and PSMD4 could be a strategy to treat MM patients with 1q21+.