Proteasome Subunit Genes Research Articles

Activating the NFE2L1-ubiquitin-proteasome system by DDI2 protects from ferroptosis.

Ferroptosis is an iron-dependent, non-apoptotic form of cell death initiated by oxidative stress and lipid peroxidation. Recent evidence has linked ferroptosis to the action of the transcription factor Nuclear factor erythroid-2 derived,-like-1 (NFE2L1). NFE2L1 regulates proteasome abundance in an adaptive fashion, maintaining protein quality control to secure cellular homeostasis, but the regulation of NFE2L1 during ferroptosis and the role of the ubiquitin-proteasome system (UPS) herein are still unclear. In the present study, using an unbiased proteomic approach charting the specific ubiquitylation sites, we show that induction of ferroptosis leads to recalibration of the UPS. RSL3-induced ferroptosis inhibits proteasome activity and leads to global hyperubiquitylation, which is linked to NFE2L1 activation. As NFE2L1 resides in the endoplasmic reticulum tethered to the membrane, it undergoes complex posttranslational modification steps to become active and induce the expression of proteasome subunit genes. We show that proteolytic cleavage of NFE2L1 by the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is a critical step for the ferroptosis-induced feed-back loop of proteasome function. Cells lacking DDI2 cannot activate NFE2L1 in response to RSL3 and show global hyperubiquitylation. Genetic or chemical induction of ferroptosis in cells with a disrupted DDI2-NFE2L1 pathway diminishes proteasomal activity and promotes cell death. Also, treating cells with the clinical drug nelfinavir, which inhibits DDI2, sensitized cells to ferroptosis. In conclusion, our results provide new insight into the importance of the UPS in ferroptosis and highlight the role of the DDI2-NFE2L1 as a potential therapeutic target. Manipulating DDI2-NFE2L1 activity through chemical inhibition might help sensitizing cells to ferroptosis, thus enhancing existing cancer therapies.

Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans.

The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction.

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.

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.

Epigenetic Control of B Cell Autoimmunity By Jmjd1c

Emerging evidence underscores the importance of e pigenetic modifications in controlling B-cell development and function. While Jmjd1c, a member of the l ysine - specific histone demethylase 3 subfamily, is known to regulate the self-renewal of normal hematopoietic stem cells and leukemia stem cells, its impact on B cell biology is unknown. To explore Jmjd1c's role in B cells, we developed B-cell-specific Jmjd1c-deficient mice through crossbreeding Jmjd1c “floxed” mice with B cell-specific Mb1-Cre mice. The absence of Jmjd1c did not impact B-cell development, but it did lead to significant alterations in basal B cell antibody production. Specifically, we observed skewed isotypes from IgG2c and IgG3 to IgG1 and IgG2b. Additionally, Jmjd1c deficiency affected antigen-specific immune responses upon T-dependent and T-independent antigen challenges, implying a crucial role of Jmjd1c in B cell function. Furthermore, auto-antibody array assays revealed increased self-reactive antibody production in Jmjd1c-deficient animals, indicating the role of Jmjd1c in regulating self-reactive B cells. At a cellular level, Jmjd1c-deficient B cells showed elevated expression of CD86, a canonical marker of B cell activation. Moreover, these B cells exhibited increased BCR-induced cell proliferation, suggesting hyperactivity to antigens, including self-antigens. Notably, Jmjd1c deficiency induced systemic autoimmune disorder in a BM12-induced systemic lupus erythematosus (SLE) animal model, resulting in amplified auto-antibody production and CD86 expression. To investigate the underlying molecular mechanisms, RNA sequencing analysis demonstrated upregulation of B cell receptor signaling related genes, NF-κB pathway signature genes, and cell cycle-related genes in Jmjd1c-deficient B cells upon anti-IgM stimulation, consistent with their hyperresponsiveness. Of particular interest, we observed upregulation of genes encoding the 26S proteasome complex, a pivotal regulator of diverse cellular processes including NF-κB pathway activation and self-antigen presentation. To explore the epigenetic aspect of these genes, we employed CUT&Tag sequencing and found that Jmjd1c deficiency correlated with increased levels of H3K36me1, a marker of active chromatin and gene transcription. Moreover, the H3K36me1 levels of B cell receptor signaling related genes, NF-κB pathway signature genes, and cell cycle-related genes were significantly augmented in Jmjd1c-deficient B cells upon anti-IgM stimulation. Notably, 26S proteasome subunit genes that were upregulated in Jmjd1c-deficient B cells exhibited higher H3K36me1 modification at the promoter regions. Collectively, our study provides valuable insights into the epigenetic control of B cell autoimmunity through the regulation of H3K36 mono-methylation by Jmjd1c. This mechanism plays a critical role in B cell activation and function, including the regulation of key genes such as those encoding the 26S proteasome complex.

Effects of Hydrogen Sulfide Donor GYY4137 on the Proteasome Pool of Colon Cancer Cells

Cancer cells are characterized by an increased level of metabolism and are highly dependent on the correct functioning of the processes that ensure homeostasis. Reactive sulfur species (RSS) are important molecular modulators of metabolic processes in both healthy and tumor cells. The effect of RSS and, in particular, H2S, on key cellular systems, including the ubiquitin-proteasome system (UPS), which ensures the destruction of most intracellular proteins, has been shown. The main components of the UPS are proteasomes ‒ multisubunit protein complexes, within which protein proteolysis occurs. At the same time, data on the effect of H2S directly on the pool of proteasomes in tumor cells are insufficient. Here, we studied the effect of incubation of SW620B8-mCherry colon adenocarcinoma cells expressing a fluorescently labeled proteasome subunit with 50, 100, and 200 µM of hydrogen sulfide donor GYY4137. The effect of the substance on the proteasome pool was assessed 6, 24, 48, and 72 h after administration. It was shown that the chymotrypsin-like and caspase-like proteasome activity decreases in cells incubated with 200 µM of the GYY4137 for 24 h. This coincided with an increase in the expression of proteasome subunit genes. In lysates of cells incubated with 200 µM GYY4137 for 48 h an increase in the content of the constitutive β5 subunit was observed. Against this background, the activity of proteasomes in cells levels off. Following prolonged incubation with GYY4137 (72h), an increase in the expression levels of some proteasome genes was also observed, though this did not have a significant effect on the activity and subunit composition of proteasomes. Thus, the data obtained indicate the modulation of proteasome activity by the hydrogen sulfide donor, as well as the effect of GYY4137 on the levels of transcription and translation of proteasome genes.

Changes in the Activity and Content of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes differing, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer’s disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (non-constitutive proteasomes), was shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and protein content in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). In samples from 5xFAD mice, at the age of 380 days, compared with samples from mice of 60 days of age, 4 and 6 times more gene transcripts of the immune subunits PSMB9 and PSMB8 were detected, as well as a significant increase in the number of immune β-subunits (2.8 times – β1i, 2.2 times – β2i) was observed. The results obtained indicate activation of the synthesis of immune subunits and assembly of non-constitutive proteasomes at the terminal stage of pathology development. At the same time, the results of electrophoresis in native conditions indicate the activation of both 20S and 26S proteasomes containing immune subunits in samples from 5xFAD mice, 380 days of age. The obtained data, in combination with available literature, indicate that the activation of non-constitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, that may reflect both the development of neuroinflammation and adaptive processes in tissues.

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.

Sequence and expression analysis of the spermatogenesis-specific gene cognates, wampa and Prosα6T, in Drosophila suzukii.

The sterile insect technique (SIT) is a highly effective biologically-based method for the population suppression of highly invasive insect pests of medical and agricultural importance. The efficacy of SIT could be significantly enhanced, however, by improved methods of male sterilization that avoid the fitness costs of irradiation. An alternative sterilization method is possible by gene-editing that targets genes essential for sperm maturation and motility, rendering them nonfunctional, similar to the CRISPR-Cas9 targeting of β2-tubulin in the genetic model system, Drosophila melanogaster. However, since genetic strategies for sterility are susceptible to breakdown or resistance in mass-reared populations, alternative targets for sterility are important for redundancy or strain replacement. Here we have identified and characterized the sequence and transcriptional expression of two genes in a Florida strain of Drosophila suzukii, that are cognates of the D. melanogaster spermatocyte-specific genes wampa and Prosalpha6T. Wampa encodes a coiled-coil dynein subunit required for axonemal assembly, and the proteasome subunit gene, Prosalpha6T, is required for spermatid individualization and nuclear maturation. The reading frames of these genes differed from their NCBI database entries derived from a D. suzukii California strain by 44 and 8 nucleotide substitutions/polymorphisms, respectively, though all substitutions were synonymous resulting in identical peptide sequences. Expression of both genes is predominant in the male testis, and they share similar transcriptional profiles in adult males with β2-tubulin. Their amino acid sequences are highly conserved in dipteran species, including pest species subject to SIT control, supporting their potential use in targeted male sterilization strategies.

Changes in the Activities and Contents of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer's disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune β-subunits (by 2.8 folds, β1i; 2.2 folds, β2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.

VDAC genes down-regulation in brain samples of individuals with schizophrenia is revealed by a systematic meta-analysis

Mitochondrial dysfunction was shown to be involved in schizophrenia pathophysiology. Abnormal energy states can lead to alterations in neural function and thereby to the cognitive and behavioral aberrations characteristics of schizophrenia. Voltage-dependent anion-selective channels (VDAC) are located in the outer mitochondrial membrane and are involved in mitochondrial energy production. Only few studies explored VDAC genes’ expression in schizophrenia, and their results were not consistent. We conducted a systematic meta-analysis of ten brain samples gene expression datasets (overall 368 samples, 179 schizophrenia, 189 controls). In addition, we conducted a meta-analysis of three blood samples datasets (overall 300 samples, 167 schizophrenia, 133 controls). Pairwise correlation analysis was conducted between the VDAC and proteasome subunit genes’ expression patterns. VDAC1, VDAC2 and VDAC3 showed significant down-regulation in brain samples of patients with schizophrenia. They also showed significant positive correlations with the proteasome subunit genes’ expression levels. Our findings suggest that VDAC genes might play a role in mitochondrial dysfunction in schizophrenia. VDAC1 was down-regulated also in blood samples, which suggests its potential role as a biomarker for schizophrenia. The correlation with proteasome subunits, which were previously shown to be down-regulated in a subgroup of the patients, suggests that our findings might characterize a subgroup of the patients. This direction has the potential to lead to patients’ stratification and more precisely-targeted therapy and necessitates further study.

Genomic Analysis of the Proteasome Subunit Gene Family and Their Response to High Density and Saline-Alkali Stresses in Grass Carp

The proteasome is a highly conserved polycatalytic enzyme that is required for cellular processes and is widely present in the nucleus and cytoplasm of archaea, as well as all eukaryotes. A total of 22 members of the proteasome subunit (CiPS) gene family were identified and characterized by scanning the grass carp (Ctenopharyngodon idella) genome. These genes were classified into two subfamilies, CiPSA and CiPSB, based on phylogenetic analysis, which was consistent with the results from other species. We examined the response of this gene family to high density and saline-alkali stresses in aquaculture using publicly available transcriptome data resources. In grass carp, CiPS member transcripts were detected in all tested tissues, with the highest expression level in the head kidney and the lowest in the liver. According to transcriptome-based expression analysis, CiPS genes play a role in response to environmental stresses in grass carp, mainly in the form of negative regulation. Interestingly, a cluster of members belonging to the CiPSB subfamily on a 15 kb region on chromosome segment CI01000319, including CiPSB8, 9, 9b, and 10, showed marked responses to high density and saline-alkali stress. It appears that CiPS genes confer stress tolerance through the regulation of common genes, as well as specific genes. In summary, our genome-wide characterization, evolutionary, and transcriptomic analysis of CiPS genes in grass carp provides valuable information for characterizing the molecular functions of these genes and utilizing them to improve stress tolerance in aquaculture.

Regulation and Functions of the ER-Associated Nrf1 Transcription Factor.

Nrf1 is a member of the nuclear erythroid 2-like family of transcription factors that regulate stress-responsive gene expression in animals. Newly synthesized Nrf1 is targeted to the endoplasmic reticulum (ER) where it is N-glycosylated. N-glycosylated Nrf1 is trafficked to the cytosol by the ER-associated degradation (ERAD) machinery and is subject to rapid proteasomal degradation. When proteasome function is impaired, Nrf1 escapes degradation and undergoes proteolytic cleavage and deglycosylation. Deglycosylation results in deamidation of N-glycosylated asparagine residues to edit the protein sequence encoded by the genome. This truncated and "sequence-edited" form of Nrf1 enters the nucleus where it induces up-regulation of proteasome subunit genes. Thus, Nrf1 drives compensatory proteasome biogenesis in cells exposed to proteasome inhibitor drugs and other proteotoxic insults. In addition to its role in proteasome homeostasis, Nrf1 is implicated in responses to oxidative stress, and maintaining lipid and cholesterol homeostasis. Here, we describe the conserved and complex mechanism by which Nrf1 is regulated and highlight emerging evidence linking this unusual transcription factor to development, aging, and disease.

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

Heterozygous missense variant of the proteasome subunit \u03b2-type 9 causes neonatal-onset autoinflammation and immunodeficiency

Impaired proteasome activity due to genetic variants of certain subunits might lead to proteasome-associated autoinflammatory syndromes (PRAAS). Here we report a de novo heterozygous missense variant of the PSMB9 proteasome subunit gene in two unrelated Japanese infants resulting in amino acid substitution of the glycine (G) by aspartic acid (D) at position 156 of the encoded protein β1i. In addition to PRAAS-like manifestations, these individuals suffer from pulmonary hypertension and immunodeficiency, which are distinct from typical PRAAS symptoms. The missense variant results in impaired immunoproteasome maturation and activity, yet ubiquitin accumulation is hardly detectable in the patients. A mouse model of the heterozygous human genetic variant (Psmb9G156D/+) recapitulates the proteasome defects and the immunodeficiency phenotype of patients. Structurally, PSMB9 G156D interferes with the β-ring-βring interaction of the wild type protein that is necessary for 20S proteasome formation. We propose the term, proteasome-associated autoinflammatory syndrome with immunodeficiency (PRAAS-ID), to indicate a separate category of autoinflammatory diseases, similar to, but distinct from PRAAS, that describes the patients in this study.

P-092: Multiple Myeloma cells depend on the DDI2/NRF1-mediated proteasome stress response for survival

<h3>Background</h3> Multiple myeloma (MM) cells depend on the ubiquitin-proteasome system for survival. Proteasome inhibitors (PIs) are FDA-approved in MM and have radically improved patient survival. However, acquisition of resistance is inevitable. The proteasome stress response (PSR) contributes to proteostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency, it is deglycosylated by NGLY1, cleaved by the aspartic protease DDI2, and translocates to the nucleus, to induce proteasome subunit gene transcription. Pharmacological targeting of NGLY1 was recently shown to sensitize cells to PIs. <h3>Hypothesis</h3> We hypothesize that blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM and represent an innovative therapeutic strategy to overcome PI resistance. <h3>Methods</h3> We used MM cell lines with distinct baseline sensitivity to PIs, including AMO1-VR, an isogenic AMO1 cell line adapted to grow in continuous bortezomib. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2/NRF1. Cell viability or tumor growth of DDI2/NRF1 KO versus non-targeting gRNA-edited cells were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate. Proteasome subunit PSMA7, PSMB5, PSMB6 and PSMD11 transcription was evaluated via real time PCR. <h3>Results</h3> Full length and cleaved NRF1 is detectable in MM cell lines and positively correlates with polyubiquitinated proteins, suggesting constitutively active PSR in MM. DDI2/NRF1 KO is cytotoxic alone or in combination with PI carfilzomib in MM cells with distinct PI sensitivity, including de-novo PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NSG mice and results in prolongation of animal survival. DDI2 KO in AMO1-VR sensitizes to carfilzomib. Further, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome subunit transcription and CT-L proteasome activity in baseline conditions and following carfilzomib treatment, resulting in increased sensitivity to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. <h3>Conclusions</h3> MM cells exhibit baseline activation of NRF1 and are dependent upon DDI2 for survival. DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome subunit biogenesis and recovery of CT-L proteasome activity, thereby increasing sensitivity to PI. Add-back of wild-type, but not of catalytically-dead DDI2 fully rescues these phenotypes. Our study provides the preclinical rationale for development of novel therapeutics targeting DDI2/NRF1 in MM.

Oleuropein Activates Neonatal Neocortical Proteasomes, but Proteasome Gene Targeting by AAV9 Is Variable in a Clinically Relevant Piglet Model of Brain Hypoxia-Ischemia and Hypothermia.

Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically relevant neonatal piglet model. Protecting and activating proteasomes could be an adjunct therapy to hypothermia. We investigated whether chymotrypsin-like proteasome activity differs regionally and developmentally in the neonatal brain. We also tested whether neonatal brain proteasomes can be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by targeting a proteasome subunit gene using recombinant adeno-associated virus-9 (AAV). During post-HI hypothermia, we treated piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ using AAV-PA28γ with green fluorescent protein (GFP). Neonatal neocortex and subcortical white matter had greater proteasome activity than did liver and kidney. Neonatal white matter had higher proteasome activity than did juvenile white matter. Lower arterial pH 1 h after HI correlated with greater subsequent cortical proteasome activity. With increasing brain homogenate protein input into the assay, the initial proteasome activity increased only among shams, whereas HI increased total kinetic proteasome activity. OLE increased the initial neocortical proteasome activity after hypothermia. AAV drove GFP expression, and white matter PA28γ levels correlated with proteasome activity and subunit levels. However, AAV proteasome modulation varied. Thus, neonatal neocortical proteasomes can be pharmacologically activated. HI slows the initial proteasome performance, but then augments ongoing catalytic activity. AAV-mediated genetic manipulation in the piglet brain holds promise, though proteasome gene targeting requires further development.

Abstract LB110: Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival

Abstract Introduction: We previously showed that an imbalance between polyubiquitinated proteins awaiting proteasomal degradation and proteasome activity is a main determinant of proteasome inhibitor (PI) sensitivity in MM. While PIs have radically improved survival of MM patients, resistance is inevitable over time and understanding the molecular mechanisms underlying it is critically important to develop novel therapies to improve patient outcomes. The proteasome stress response (PSR) is an evolutionarily conserved homeostatic mechanism triggered by insufficient proteasome activity and aimed at restoring homeostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency is cleaved by the aspartic protease DDI2, translocates to the nucleus, and induces proteasome subunit gene transcription. It is unknown whether blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM. Materials and methods: We used human MM cell lines with distinct baseline sensitivity to PIs. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2 and NRF1. Cell viability or tumor growth of DDI2/NRF1 KO cells versus cells edited with a non-targeting gRNA were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate upon incubation with protein lysates from DDI2 WT, KO, or addback cells. Results: We detected full-length and cleaved NRF1 in human MM cell lines, but not other hematologic cancer cell lines, suggesting constitutively active PSR in MM. Query of the dependency map (depmap.org) shows that MM is the cell line type most highly dependent on DDI2. Consistently, DDI2 or NRF1 KO is cytotoxic alone or in combination with PI across a panel of MM cells with distinct PI sensitivity, including de novo, PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NOD SCID mice and results in prolongation of animal survival. While biallelic DDI2 or NRF1 KO monoclones could not be generated across 7 distinct MM cell lines, consistent with these genes being essential, we successfully established DDI2 KO monoclones in AMO1-VR, an isogenic AMO1 cell line variant adapted to grow in continuous bortezomib. Even in this cell line model of acquired PI resistance, DDI2 KO sensitized cells to PI carfilzomib. In this model, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome recovery after irreversible PI treatment and sensitizing to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. Conclusions: Our data show that MM cells are intrinsically dependent on an intact DDI2/NRF1 axis to survive in baseline conditions and upon proteasomal inhibition. We propose that DDI2 is an unexplored, promising molecular target for MM treatment by disrupting the PSR and exacerbating proteotoxicity. This study provides the preclinical rationale for the development of novel therapeutics targeting the DDI2/NRF1 pathway to overcome acquired and de novo PI resistance and improve patient outcomes in MM. Citation Format: Tianzeng Chen, Matthew Ho, Jenna Briere, Maria Moscvin, Peter G. Czarnecki, Kenneth C. Anderson, T. Keith Blackwell, Giada Bianchi. Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB110.

1,4-dihydropyridine derivatives increase mRNA expression of Psma3, Psmb5, and Psmc6 in rats.

The ubiquitin-proteasome system modifies different cellular and protein functions. Its dysregulation may lead to disrupted proteostasis associated with multiple pathologies and aging. Pharmacological regulation of proteasome functions is already an important part of the treatment of several diseases. 1,4-dihydropyridine (1,4-DHP) derivatives possess different pharmacological activities, including antiaging and neuroprotective. The aim of this study was to investigate the effects of several 1,4-DHP derivatives on mRNA expression levels of proteasomal genes Psma3, Psmb5, and Psmc6 in several organs of rats. Rats were treated with metcarbatone, etcarbatone, glutapyrone, styrylcarbatone, AV-153-Na, or AV-153-Ca per os for three days. mRNA expression levels were determined with real-time polymerase chain reaction (PCR). For AV-153-Na and AV-153-Ca, we also determined the expression of the Psma6 gene. In the kidney, metcarbatone, etcarbatone, styrylcarbatone, and AV-153-Na increased the expression of all analysed genes. Glutapyrone increased the expression of Psmb5 and Psmc6 but did not affect the expression of Psma3. In the blood, glutapyrone increased Psmb5 expression. In the liver, AV-153-Na increased the expression of Psma6 and Psmc6 but lowered the expression of Psmb5, while AV-153-Ca only increased Psma6 expression. The ability of 1,4-DHP derivatives to increase the expression of proteasome subunit genes might hold a therapeutic potential in conditions associated with impaired proteasomal functions, but further research is needed.

Roles of CNC Transcription Factors NRF1 and NRF2 in Cancer.

Simple SummaryAlthough NRF1 (nuclear factor erythroid 2-like 1; NFE2L1) and NRF2 (nuclear factor erythroid 2-like 2; NFE2L2) belong to the CNC (cap‘n’collar) transcription factor family and share DNA recognition elements, their functions in vivo are substantially different. In cancer cells, while NRF2 confers therapeutic resistance via increasing antioxidant capacity and modulating glucose and glutamine metabolism, NRF1 confers therapeutic resistance via triggering proteasome bounce back response. Proteasome inhibition activates NRF1, and NRF1, in turn, activates the proteasome by inducing the transcriptional activation of proteasome subunit genes. One of the oncometabolites, UDP-GlcNAc (uridine diphosphate N-acetylglucosamine), has been found to be a key to the NRF1-mediated proteasome bounce back response. In this review, we introduce the roles of NRF1 in the cancer malignancy in comparison with NRF2. Cancer cells exhibit unique metabolic features and take advantage of them to enhance their survival and proliferation. While the activation of NRF2 (nuclear factor erythroid 2-like 2; NFE2L2), a CNC (cap‘n’collar) family transcription factor, is effective for the prevention and alleviation of various diseases, NRF2 contributes to cancer malignancy by promoting aggressive tumorigenesis and conferring therapeutic resistance. NRF2-mediated metabolic reprogramming and increased antioxidant capacity underlie the malignant behaviors of NRF2-activated cancer cells. Another member of the CNC family, NRF1, plays a key role in the therapeutic resistance of cancers. Since NRF1 maintains proteasome activity by inducing proteasome subunit genes in response to proteasome inhibitors, NRF1 protects cancer cells from proteotoxicity induced by anticancer proteasome inhibitors. An important metabolite that activates NRF1 is UDP-GlcNAc (uridine diphosphate N-acetylglucosamine), which is abundantly generated in many cancer cells from glucose and glutamine via the hexosamine pathway. Thus, the metabolic signatures of cancer cells are closely related to the oncogenic and tumor-promoting functions of CNC family members. In this review, we provide a brief overview of NRF2-mediated cancer malignancy and elaborate on NRF1-mediated drug resistance affected by an oncometabolite UDP-GlcNAc.