Proteasome Subunit Research Articles

Mutant dominant-negative rhodopsin∆I256 causes protein aggregates degraded via ERAD and prevents normal rhodopsin from proper membrane trafficking.

Dominant mutations in the rhodopsin gene (Rho) contribute to 25% of autosomal dominant retinitis pigmentosa (adRP), characterized by photoreceptor loss and progressive blindness. One such mutation, Rho ∆I256 , carries a 3-bp deletion, resulting in the loss of one of two isoleucines at codons 255 and 256. Our investigation, using recombinant expression in HEK293 and COS-7 cells, revealed that Rho ∆I256, akin to the known adRP mutation Rho P23H, induces the formation of rhodopsin protein (RHO) aggregates at the perinuclear region. Co-expression of Rho ∆I256 or Rho P23H with wild-type Rho WT, mimicking the heterozygous genotype of adRP patients, demonstrated the dominant-negative effect, as all isoforms were retained in perinuclear aggregates, impeding membrane trafficking. In retinal explants from WT mice, mislocalization of labeled adRP isoforms at the outer nuclear layer was observed. Further analysis revealed that RHO∆I256 aggregates are retained at the endoplasmic reticulum (ER), undergo ER-associated degradation (ERAD), and colocalize with the AAA-ATPase escort chaperone valosin-containing protein (VCP). These aggregates are polyubiquitinated and partially colocalized with the 20S proteasome subunit beta-5 (PSMB5). Pharmacological inhibition of proteasome- or VCP activity increased RHO∆I256 aggregate size. In summary, RHO∆I256 exhibits dominant pathogenicity by sequestering normal RHOWT in ER aggregates, preventing its membrane trafficking and following the ERAD degradation.

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.

Modulation of proteasome subunit selectivity of syringolins

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure–activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure–activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding β5 and β2 subunits.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Proteostatic Remodeling of Small Heat Shock Chaperones─Crystallins by Ran-Binding Protein 2─and the Peptidyl-Prolyl cis-trans Isomerase and Chaperone Activities of Its Cyclophilin Domain.

Disturbances in protein phase transitions promote protein aggregation─a neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also regulate phase transitions and proteostasis by suppressing protein aggregation. Ranbp2 haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against phototoxicity by proteostatic regulations of neuroprotective substrates of Ranbp2 and by suppressing the buildup of polyubiquitylated substrates. Losses of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 recapitulate molecular effects of Ranbp2 haploinsufficiency. These CY impairments also stimulate deubiquitylation activities and phase transitions of 19S cap subunits of the 26S proteasome that associates with Ranbp2. However, links between CY moonlighting activity, substrate ubiquitylation, and proteostasis remain incomplete. Here, we reveal the Ranbp2 regulation of small heat shock chaperones─crystallins in the chorioretina by proteomics of mice with total or selective modular deficits of Ranbp2. Specifically, loss of CY PPIase of Ranbp2 upregulates αA-Crystallin, which is repressed in adult nonlenticular tissues. Conversely, impairment of CY's chaperone activity opposite to the PPIase pocket downregulates a subset of αA-Crystallin's substrates, γ-crystallins. These CY-dependent effects cause age-dependent and chorioretinal-selective declines of ubiquitylated substrates without affecting the chorioretinal morphology. A model emerges whereby inhibition of Ranbp2's CY PPIase remodels crystallins' expressions, subdues molecular aging, and preordains the chorioretina to neuroprotection by augmenting the chaperone capacity and the degradation of polyubiquitylated substrates against proteostatic impairments. Further, the druggable Ranbp2 CY holds pan-therapeutic potential against proteotoxicity and neurodegeneration.

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.

Pre-coating cRGD-modified bovine serum albumin enhanced the anti-tumor angiogenesis of siVEGF-loaded chitosan-based nanoparticles by manipulating the protein corona composition

Chitosan-based nanoparticles inevitably adsorb numerous proteins in the bloodstream, forming a protein corona that significantly influences their functionality. This study employed a pre-coated protein corona using cyclic Arg-Gly-Asp peptide (cRGD)-modified bovine serum albumin (BcR) to confer tumor-targeting capabilities on siVEGF-loaded chitosan-based nanoparticles (CsR/siVEGF NPs) and actively manipulated the serum protein corona composition to enhance their anti-tumor angiogenesis. Consequently, BcR effectively binds to the nanoparticles' surface, generating nanocarriers of appropriate size and stability that enhance the inhibition of endothelial cell proliferation, migration, invasion, and tube formation, as well as suppress tumor proliferation and angiogenesis in tumor-bearing nude mice. Proteomic analysis indicated a significant enrichment of serotransferrin, albumin, and proteasome subunit alpha type-1 in the protein corona of BcR-precoated NPs formed in the serum of tumor-bearing nude mice. Additionally, there was a decrease in proteins associated with complement activation, immunoglobulins, blood coagulation, and acute-phase responses. This modification resulted in an enhanced impact on anti-tumor angiogenesis, along with a reduction in opsonization and inflammatory responses. Therefore, pre-coating of nanoparticles with a functionalized albumin corona to manipulate the composition of serum protein corona emerges as an innovative approach to improve the delivery effectiveness of chitosan-based carriers for siVEGF, targeting the inhibition of tumor angiogenesis.

Proteasome-Associated Syndromes: Updates on Genetics, Clinical Manifestations, Pathogenesis, and Treatment.

The ubiquitin-proteasome system (UPS) has a critical role in post-translational protein modification that is essential for the maintenance of all cellular functions, including immune responses. The proteasome complex is ubiquitously expressed and is responsible for degradation of short-lived structurally abnormal, misfolded and not-needed proteins that are targeted for degradation via ubiquitin conjugation. Over the last 14years, an increasing number of human diseases have been linked to pathogenic variants in proteasome subunits and UPS regulators. Defects of the proteasome complex or its chaperons - which have a regulatory role in the assembly of the proteasome - disrupt protein clearance and cellular homeostasis, leading to immune dysregulation, severe inflammation, and neurodevelopmental disorders in humans. Proteasome-associated diseases have complex inheritance, including monogenic, digenic and oligogenic disorders and can be dominantly or recessively inherited. In this review, we summarize the current known genetic causes of proteasomal disease, and discuss the molecular pathogenesis of these conditions based on the function and cellular expression of mutated proteins in the proteasome complex.

Depletion of proteasome subunit PSMD1 induces cancer cell death via protein ubiquitination and DNA damage, irrespective of p53 status

Hepatocellular carcinoma (HCC) is characterized by high incidence and fatality rates worldwide. In our exploration of prognostic factors in HCC, the 26s proteasome subunit, non-ATPase 1 (PSMD1) protein emerged as a significant contributor, demonstrating its potential as a therapeutic target in this aggressive cancer. PSMD1 is a subunit of the 19S regulatory particle in the 26S proteasome complex; the 19S particle controls the deubiquitination of ubiquitinated proteins, which are then degraded by the proteolytic activity of the complex. Proteasome-targeting in cancer therapy has received significant attention because of its practical application as an established anticancer agent. We investigated whether PSMD1 plays a critical role in cancer owing to its prognostic significance. PSMD1 depletion induced cell cycle arrest in G2/M phase, DNA damage and apoptosis of cancer cells, irrespective of the p53 status. PSMD1 depletion-mediated cell death was accompanied by an increase in overall protein ubiquitination. These phenotypes occurred exclusively in cancer cells, with no effects observed in normal cells. These findings indicate that PSMD1 depletion-mediated ubiquitination of cellular proteins induces cell cycle arrest and eventual death in cancer cells, emphasizing PSMD1 as a potential therapeutic target in HCC.

PSMA2 promotes glioma proliferation and migration via EMT

BackgroundGliomas advance rapidly and are associated with a poor prognosis. Epithelial-mesenchymal transition (EMT) accelerates the progression of gliomas, exerting a pivotal role in glioma development. Proteasome subunit alpha type-2 (PSMA2) exhibits high expression levels in gliomas. however, its specific involvement in glioma progression and its correlation with EMT remain elusive. This study aims to elucidate the role of PSMA2 in glioma progression and its potential association with EMT. MethodsOnline tools were employed to analyze the expression patterns and survival curves of PSMA2 in gliomas. The relationship between PSMA2 and various characteristics of glioma patients was investigated using data from the TCGA and CGGA databases. In vitro, cell proliferation and migration were assessed through CCK-8, colony formation, and transwell assays. Furthermore, a tumor xenograft model in nude mice was established to evaluate in vivo tumorigenesis. Protein binding to PSMA2 was scrutinized using co-immunoprecipitation MS (co-IP MS). The potential biological functions and molecular pathways associated with PSMA2 were explored through GO analysis and KEGG analysis, and the correlation between PSMA2 and EMT was validated through correlation analysis and Western blot experiments. ResultsBioinformatics analysis revealed a significant upregulation of PSMA2 across various cancers, with particularly heightened expression in gliomas. Moreover, elevated PSMA2 levels were correlated with advanced tumor stages and diminished survival rates among glioma patients. Inhibition of PSMA2 demonstrated a pronounced suppressive effect on glioma cell proliferation, both in vitro and in vivo. Knockdown of PSMA2 also impeded the migratory capacity of glioma cells. GO and KEGG enrichment analyses indicated that PSMA2-binding proteins (identified through Co-IP-MS) were associated with cell adhesion molecule binding and cadherin binding. Western blot results further confirmed the role of PSMA2 in promoting epithelial-mesenchymal transition (EMT) in glioma cells. ConclusionOur study provides evidence supporting the role of PSMA2 as a regulatory factor in EMT and suggests its potential as a prognostic biomarker for glioma progression.

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.

Abstract 7344: Role of ubiquitin receptor ADRM1 (Rpn13) in prostate cancer

Abstract Adhesion-regulating molecule 1 (ADRM1)/Rpn13 is a component of the 19S regulatory subunit of the 26S proteasome [1]. ADRM1 plays a crucial role in the recognition of polyubiquitinated substrates of the proteasome and their subsequent deubiquitination and degradation [2]. ADRM1 transcript levels are commonly upregulated in diverse tumor types [3]. The androgen receptor (AR) plays a predominant role in prostate cancer (PCa) pathology. In this study, we investigated the expression of ADRM1/Rpn13 in different stages of prostate cancer and its association with AR expression. TCGA data from UALCAN was used for understanding ADRM1 expression on different stages of diseases and single-cell RNA-sequencing data of prostate cancer cell line was obtained from Prostate Cancer Atlas. TCGA (cell, 2015) data set was used for investigating the correlation between ADRM1 and AR by using R programming. Our study from the TCGA dataset reveals that ADRM1 expression increased significantly with the aggressiveness of prostate cancer (increase consistently with the Gleason score) from the TCGA dataset. We analyzed ADRM1 and AR expression in different prostate cancer cell lines and obtained that ADRM1 expressed significantly higher in prostate cancer cell lines which had lower AR expression. TCGA (cell, 2015) dataset indicated a negative correlation between ADRM1 and AR expression in prostate cancer. Our preliminary analysis indicated that ADRM1 inhibitor might be a potential therapeutic option for AR-independent prostate cancer. Citation Format: Shahjalal Chowdhury, Honghe Wang, Chitra Nayak, Balasubramanyam Karanam. Role of ubiquitin receptor ADRM1 (Rpn13) in prostate cancer [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 7344.

Abstract 4316: Investigation the role of DDI2 in autophagy

Abstract Background: The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are critical intracellular protein degradation pathways. In response to proteasome dysfunction, cells initiate compensatory autophagy to manage proteotoxic stress, which impacts cancer therapy efficacy. Despite current insights, the exact molecular mechanisms underlying the induction of autophagy due to impaired proteasome function remain unclear. The transcription factor nuclear factor erythroid-2-related factor 1 (NRF1) is the master regulator of proteasome subunit genes, which is activated by the protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome inhibition. DDI2 also acts as an ubiquitin shuttling factor, and its depletion causes the accumulation of highly ubiquitinated proteins, resulting in increased sensitivity to proteasome inhibition in cancer cells. However, cancer cells might trigger compensatory autophagy in response to DDI2 depletion to degrade ubiquitinated protein aggregates. Here, we demonstrated that DDI2 depletion plays a role in autophagy induction. Methods: A panel of cell lines of different origins (EW16 and ES1 human Ewing sarcoma, MIA PaCa-2 human pancreatic cancer, and NIH-3T3 murine fibroblasts) either control or DDI2-deficient were examined for autophagy activity following treatment with the autophagy inhibitor chloroquine (CQ). Through total proteome analysis, we identified an increase in cellular communication network factor 1 (CCN1) protein levels in DDI2-depleted cells, which plays a role in autophagy induction. To determine whether CCN1 is required and sufficient to induce autophagy, we applied small interfering RNA (siRNA), and viral constructs to knockdown and overexpress CCN1. Results: The autophagic flux measurements in four different DDI2-deficient cell lines revealed elevated LC3B-II protein levels, a known autophagy marker, following CQ treatment compared to their control. Western blotting analysis confirmed increased CCN1 protein levels in the same cell lines. The autophagy induction observed in DDI2-deficient cells was diminished by CCN1-siRNA, demonstrating that CCN1 may be required for autophagy induction. Furthermore, overexpressing CCN1 in wild-type cells resulted in increased LC3B-II protein levels, indicating that CCN1 alone is sufficient to induce autophagy. Conclusion: Targeting autophagy is a promising therapeutic strategy to improve the effects of anti-cancer therapies. We demonstrated that upon depletion of ubiquitin shuttle factor DDI2, secretory protein CCN1 accumulates leading to the subsequent induction of autophagy. These findings improve our understanding of the molecular basis governing DDI2’s role in crosstalk between protein degradation pathways. Targeting DDI2 in combination with autophagy inhibition could further potentiate these drugs as anti-cancer therapies. Citation Format: Nayyerehalsadat Hosseini, Holly A. Byers, Senthil K. Radhakrishnan. Investigation the role of DDI2 in autophagy [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 4316.

Abstract 450: GCN2 protects cancer cells from hypertranslation and metabolic crisis

Abstract It is widely assumed that cancer cells rely on high levels of protein synthesis to support growth and proliferation. However, protein synthesis, including ribosome biogenesis, is a highly resource- and energy-demanding process that needs to be coordinated with protein degradation and metabolic programs. GCN2 is an evolutionarily conserved kinase that is primarily activated by uncharged tRNAs when amino acid levels drop and activates the Integrated Stress Response (ISR), which encompasses a global reduction in translation and increased amino acid uptake and generation. GCN2 has been shown to support cancer cell survival under conditions of nutrient scarcity. Here, we used an integrated systems biology approach to delineate the multifaceted role of GCN2 in cancer cells that do not suffer from nutrient shortages. We carried out a broad array of multiomic analyses (RNA-seq, TMT-labelling based proteomics, Ribo-seq, LC-MS metabolomics) of melanoma cells in which GCN2 was inhibited (GCN2iB) or genetically depleted (constitutive and inducible shRNA) in cell culture and in vivo in murine xenografts, supplemented by puromycinylation-based quantification of global protein synthesis. GCN2 inhibition in A375 cells triggered a transcriptome and proteome response that was dominated by the induction of processes linked to protein and ribosome synthesis while suppressing metabolic pathways such as glycolysis and the proteasome. We confirmed these observations in vivo via RNA-seq of tumor samples from mice xenografted with A375 cells in which GCN2 was knocked down by induced shRNA expression and from xenografted mice that were treated orally with GCN2iB. Ribo-seq further demonstrated that, upon GCN2 inhibition, A375 cells expand their translational machinery. Notably, we identified 50 ribosomal genes that were translated at higher rate in GCN2-inhibited cells and whose expression was regulated exclusively at the translational level. GCN2 inhibition/depletion also resulted in an approximately 1.8-fold increase in global protein synthesis and altered the druggable proteome. Moreover, metabolite profiling of GCN2iB-treated cells revealed a loss of metabolic homeostasis including substantial alterations in glycolysis and both amino acid and lipid metabolism. In addition, we observed a significant decrease in ATP levels despite increased translation of mitochondrial ETC complex proteins. Proteomic and translatomic analyses revealed a failure of cells to increase proteasomal subunits and to maintain the production of ECM proteins. Last, but not least, we did not observe a role of GCN2 in preventing or resolving ribosome collisions. Taken together, our findings reveal GCN2 as a master regulator of translation in cancer cells that do not suffer from nutrient scarcity that keeps protein synthesis in check to maintain metabolic homeostasis. Citation Format: Monica Roman-Trufero, Istvan T. Kleijn, Kevin Blighe, Paula Saavedra-Garcia, Jinglin Zhou, Abigail Gaffar, Marilena Christoforou, Mariia Yuneva, Audrey M. Michel, Glenn R. Masson, Vahid Shahrezaei, Holger W. Auner. GCN2 protects cancer cells from hypertranslation and metabolic crisis [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 450.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

Cisplatin Disrupts Proteasome Bounce-Back Effect through Suppressing ZEB1/Nfe2l1 in Cholangiocarcinoma.

Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown. Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry. Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells. Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.

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.

Person-specific differences in ubiquitin-proteasome mediated proteostasis in human neurons.

Impairment of the ubiquitin-proteasome system (UPS) has been implicated in abnormal protein accumulation in Alzheimer's disease. It remains unclear if genetic variation affects the intrinsic properties of neurons that render some individuals more vulnerable to UPS impairment. Induced pluripotent stem cell (iPSC)-derived neurons were generated from over 50 genetically variant and highly characterized participants of cohorts of aging. Proteomic profiling, proteasome activity assays, and Western blotting were employed to examine neurons at baseline and in response to UPS perturbation. Neurons with lower basal UPS activity were more vulnerable to tau accumulation following mild UPS inhibition. Chronic reduction in proteasome activity in human neurons induced compensatory elevation of regulatory proteins involved in proteostasis and several proteasome subunits. These findings reveal that genetic variation influences basal UPS activity in human neurons and differentially sensitizes them to external factors perturbing the UPS, leading to the accumulation of aggregation-prone proteins such as tau. Polygenic risk score for AD is associated with the ubiquitin-proteasome system (UPS) in neurons. Basal proteasome activity correlates with aggregation-prone protein levels in neurons. Genetic variation affects the response to proteasome inhibition in neurons. Neuronal proteasome perturbation induces an elevation in specific proteins involved in proteostasis. Low basal proteasome activity leads to enhanced tau accumulation with UPS challenge.

Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria.

Mutations in FBXO7 have been discovered to be associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerisation domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts to facilitate SCFFbxo7-mediated ubiquitination of MiD49. The L250P mutation reduces the SCFFbxo7 ligase-mediated ubiquitination of a subset of its known substrates. Although MiD49/51 expression was reduced in patient cells, there was no effect on the mitochondrial network. However, patient cells show reduced levels of mitochondrial function and mitophagy, higher levels of ROS and are less viable under stress. Our study demonstrates that Fbxo7 and PI31 regulate proteasomes and mitochondria and reveals a new function for PI31 in enhancing the SCFFbxo7 E3 ubiquitin ligase activity.