Proteasome Genes Research Articles

STAT3 protects dopaminergic neurons against degeneration in animal model of Parkinson's disease

IntroductionParkinson's disease (PD) is the most prevalent disorder of the basal ganglia, propagated by the degeneration of axon terminals within the striatum and subsequent loss of dopaminergic neurons in the substantia nigra (SN). Exposure of environmental neurotoxins and mutations of several mitochondrial and proteasomal genes are primarily responsible. MethodsTo determine whether signal transducer and activator of transcription 3 (STAT3) could protect dopaminergic neurons against degeneration, we first screened it in the in vitro capacity using immortalized rat dopaminergic N27 cells under 6-OHDA neurotoxicity. We then evaluated the effectiveness of constitutively active (ca) STAT3 as a neuroprotective agent on N27 cells in a 6-hydroxydopamine (6-OHDA) induced rat model of PD and compared it to control animals or animals where AAV/caRheb was expressed in SN. Behavioral outcomes were assessed using rotational and cylinder assays and mitochondrial function using reactive oxygen species (ROS) levels. ResultsUsing flow cytometry, the in vitro analysis determined caSTAT3 significantly decreased dopaminergic neuronal death under 6-OHDA treatment conditions. Importantly, in vivo overexpression of caSTAT3 in SN dopaminergic neurons using AAV-mediated expression demonstrated significant neuroprotection of dopaminergic neurons following 6-OHDA. Both caSTAT3 and caRheb + caSTAT3 co-injection into substantia nigra reduced D-amphetamine-induced rotational behavior and increased ipsilateral forelimb function when compared to control animals. In addition, caSTAT3 decreased mitochondrial ROS production following 6-OHDA induced neurotoxicity. ConclusioncaSTAT3 confers resistance against ROS production in mitochondria of susceptible SN dopaminergic neurons potentially offering a new avenue for treatment against PD.

Nrf1 is not a direct target gene of SREBP1, albeit both are integrated into the rapamycin-responsive regulatory network in human hepatoma cells.

The essential role of protein degradation by ubiquitin-proteasome system is exerted primarily for maintaining cellular protein homeostasis. The transcriptional activation of proteasomal genes by mTORC1 signaling depends on Nrf1, but whether this process is directly via SREBP1 remains elusive. In this study, our experiment evidence revealed that Nrf1 is not a direct target of SREBP1, although both are involved in the rapamycin-responsive regulatory networks. Closely scrutinizing two distinct transcriptomic datasets unraveled no significant changes in transcriptional expression of Nrf1 and almost all proteasomal subunits in either siSREBP2-silencing cells or SREBP1-∕-MEFs, when compared to equivalent controls. However, distinct upstream signaling to Nrf1 dislocation by p97 and its processing by DDI1/2, along with downstream proteasomal expression, may be monitored by mTOR signaling, to various certain extents, depending on distinct experimental settings in different types of cells. Our further evidence has been obtained from DDI1-∕-(DDI2insC) cells, demonstrating that putative effects of mTOR on the rapamycin-responsive signaling to Nrf1 and proteasomes may also be executed partially through a DDI1/2-independent mechanism, albeit the detailed regulatory events remain to be determined.

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.

Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging

—Proteasomes are key components of the ubiquitin–proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit β5; increased expression of genes encoding immune proteasome subunits β5i and β1i. In tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the β5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

Evaluation of the NRF1-proteasome axis as a therapeutic target in breast cancer

Proteasomes are multi-subunit complexes that specialize in protein degradation. Cancer cells exhibit a heightened dependence on proteasome activity, presumably to support their enhanced proliferation and other cancer-related characteristics. Here, a systematic analysis of TCGA breast cancer datasets revealed that proteasome subunit transcript levels are elevated in all intrinsic subtypes (luminal, HER2-enriched, and basal-like/triple-negative) when compared to normal breast tissue. Although these observations suggest a pan-breast cancer utility for proteasome inhibitors, our further experiments with breast cancer cell lines and patient-derived xenografts (PDX) pointed to triple-negative breast cancer (TNBC) as the most sensitive subtype to proteasome inhibition. Finally, using TNBC cells, we extended our studies to in vivo xenograft experiments. Our previous work has firmly established a cytoprotective role for the transcription factor NRF1 via its ability to upregulate proteasome genes in response to proteasome inhibition. In further support of this notion, we show here that NRF1 depletion significantly reduced tumor burden in an MDA-MB-231 TNBC xenograft mouse model treated with carfilzomib. Taken together, our results point to TNBC as a particularly vulnerable breast cancer subtype to proteasome inhibition and provide a proof-of-principle for targeting NRF1 as a viable means to increase the efficacy of proteasome inhibitors in TNBC tumors.

The Genetic Diversity of Proteasome Genes in the T1DM Polish Population.

In the present study, the genetic variations in PSMB5 (rs11543947), PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) UPS gene cluster was investigated in type 1 diabetes and healthy donors in the Polish population. The study comprised 105 patients with type 1 diabetes mellitus (T1DM) and 214 controls. All were genotyped by PCR and restriction digestion analysis or Sanger sequencing. Rs1048990 and rs2348071 were found to be neutral to T1DM (p-value: 0.499 and 0.656, respectively). According to the multiple loci genotype (MLG) analysis, the major homozygote of the tested polymorphisms had a protective effect. The most common MLG in the T1DM group was characterised by simultaneous risk factors at rs11543947, rs2277460, rs2295826 and rs2295827 (pvalue: <0.0001 vs. MGL1). Multiple locus haplotype analysis revealed a similar dependence, with common alleles at all tested loci demonstrating a protective effect, and the rare alleles increasing T1DM risk (p-value: <0.0001 vs. MLH1). Our study suggests that the proteasome gene polymorphisms rs11543947, rs2277460, rs2295826, and rs2295827 could be potential markers for T1DM susceptibility in the Polish population.

The transcription factor NRF1 (NFE2L1) activates aggrephagy by inducing p62 and GABARAPL1 after proteasome inhibition to maintain proteostasis

The ubiquitin‒proteasome system (UPS) and autophagy are the two primary cellular pathways of misfolded or damaged protein degradation that maintain cellular proteostasis. When the proteasome is dysfunctional, cells compensate for impaired protein clearance by activating aggrephagy, a type of selective autophagy, to eliminate ubiquitinated protein aggregates; however, the molecular mechanisms by which impaired proteasome function activates aggrephagy remain poorly understood. Here, we demonstrate that activation of aggrephagy is transcriptionally induced by the transcription factor NRF1 (NFE2L1) in response to proteasome dysfunction. Although NRF1 has been previously shown to induce the expression of proteasome genes after proteasome inhibition (i.e., the proteasome bounce-back response), our genome-wide transcriptome analyses identified autophagy-related p62/SQSTM1 and GABARAPL1 as genes directly targeted by NRF1. Intriguingly, NRF1 was also found to be indispensable for the formation of p62-positive puncta and their colocalization with ULK1 and TBK1, which play roles in p62 activation via phosphorylation. Consistently, NRF1 knockdown substantially reduced the phosphorylation rate of Ser403 in p62. Finally, NRF1 selectively upregulated the expression of GABARAPL1, an ATG8 family gene, to induce the clearance of ubiquitinated proteins. Our findings highlight the discovery of an activation mechanism underlying NRF1-mediated aggrephagy through gene regulation when proteasome activity is impaired.

Dynamic Changes in the Activity and Content of Particular Proteasome Forms in Cerebral Cortex of C57BL/6 Mice during Aging

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes were revealed, as well as an increased expression of their individual forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in the cerebral cortex samples from C57BL/6 mice at the age of 60, 190, 380, and 720 days. In addition, the content of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis under native conditions were characterized. Compared with samples from young animals, in the cerebral cortex of animals aged 720 days the following changes in the expression patterns of proteasome genes were revealed: a decrease in the expression of PSMB5 gene encoding the constitutive proteasome subunit β5; activation of genes encoding immune subunits β5i and β1i. In clarified tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, decreased chymotrypsin-like activity and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity were also revealed. Analysis of the activity of native complexes in the tissues of old animals revealed decreased chymotrypsin-like activity of both 26S and 20S proteasomes containing the β5i subunit. Based on the data obtained, it can be assumed that changes in the pool of non-constitutive proteasomes reflect aging-associated adaptive processes in mouse brain.

Identification of eight novel proteasome variants in five unrelated cases of proteasome-associated autoinflammatory syndromes (PRAAS).

Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes, PSMB8 and PSMB10, whereas one patient showed additive loss-of-function mutations in PSMB8. Variants in two previously not associated proteasome genes, PSMA5 and PSMC5, were found in a patient who also carried the PSMB8 founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.

A bioinformatic investigation of proteasome and autophagy expression in the central nervous system

The ubiquitin proteasome system (UPS) and autophagy lysosome pathway (ALP) are crucial in the control of protein quality. However, data regarding the relative significance of UPS and ALP in the central nervous system (CNS) are limited. In the present study, using publicly available data, we computed the quantitative expression status of UPS- and ALP-related genes and their products in the CNS as compared with that in other tissues and cells. We obtained human and mouse gene expression datasets from the reference expression dataset (RefEx) and Genevestigator (a tool for handling curated transcriptomic data from public repositories) as well as human proteomics data from the proteomics database (ProteomicsDB). The expression levels of genes and proteins in four categories—ubiquitin, proteasome, autophagy, and lysosome––in the cells and tissues were assessed. Perturbation of the gene expression by drugs was also analyzed for the four categories. Compared with that for ubiquitin, autophagy, and lysosome, gene expression for proteasome was consistently lower in the CNS of mice but was more pronounced in humans. Neural stem cells and neurons showed low proteasome gene expression as compared with embryonic stem cells. Proteomic analyses, however, did not show trends similar to those observed in the gene expression analyses. Perturbation analyses revealed that azithromycin and vitamin D3 upregulated the expression of both UPS and ALP. Gene and proteomic expression data could offer a fresh perspective on CNS pathophysiology. Our results indicate that disproportional expression of UPS and ALP might affect CNS disorders and that this imbalance might be redressed by several therapeutic candidates.

Analytical screening of polymorphic variants of 20S proteasome genes when planning a study of pathogenetic effects of modification of NFKB1 post-translational processing

Aim. Formation of polymorphic variants panel of the proteasome genes 20S, potentially significant for the study as balance modifier factors of p105/p50 NFKB1.Materials and methods. Determination of genes that encode proteins of the multisubunit proteasome complex prospective for research purposes, was carried out on the basis of information retrieved from eLIBRARY and PubMed. The source of information for the formation of polymorphic variants panel of genes (SNP, single nucleotide polymorphism) was the Ensembl genomic browser, http://www.ensembl.org. The structure of genes is described by the NCBI (databases Gene, http:// www.ncbi.nlm.nih.gov/gene). The panel was filled with the minor allelic frequency in the population (MAF), the localization of SNP in the gene structure and the availability of data on the relationship with multifactorial diseases and other effects in mind. To calculate the genetic distances between populations, we used the methord of comparing the populations by frequencies of polymorphic marker alleles proposed by Ney, the obtained matrices are illustrated by the method of multidimensional scaling in space using Statistica v.8.0.Results. Discussion of the algorithm and results of analytical screening of polymorphic variants of 14 genes (PSMA1-PSMA7, PSMB1–PSMB7) encoding proteasome subunits 20S. The characteristics of the SNP panel are given, compiled with the selection criteria taken into account. According to the data on the frequencies of polymorphic gene variants, the features of global and European population gene pools (283 SNP), as well as samples from Russian populations (20 SNP) are analyzed. Based on the results of the analysis of information on the associations of selected SNPs with various diseases, a panel (42 SNPs) of 20S proteasome genes was formed, potentially significant for the study as factors modifying the p105/p50 NFKB1 balance.Conclusion. Annotation of the formed panel of SNP genes of the 20S proteasome with MAF&gt;0.1 indicates the potential role of polymorphism in the pathogenesis of diseases of various profiles. This may be of research interest to the formed panel in context of implementation of traditional approaches – the search for candidate genes based on the analysis of associations with diseases, as well as the analysis of the influence of SNP on the level of genetic expression, synthesis of gene products, NFKB1 processing and p105/p50 balance in silico and on model objects.

NFE2L3 drives hepatocellular carcinoma cell proliferation by regulating the proteasome-dependent degradation of ISGylated p53.

Nuclear factor erythroid 2-like 3 (NFE2L3) is a member of the cap 'n' collar basic-region leucine zipper (CNC-bZIP) transcription factor family that plays a vital role in modulating oxidation-reduction steady-state and proteolysis. Accumulating evidence suggests that NFE2L3 participates in cancer development; however, little is known about the mechanism by which NFE2L3 regulates hepatocellular carcinoma (HCC) cell growth. Here, we confirmed that NFE2L3 promotes HCC cell proliferation by acting as a transcription factor, which directly induces the expression of proteasome and interferon-stimulated gene 15 (ISG15) to enhance the proteasome-dependent degradation of ISGylated p53. Post-translational ISGylation abated the stability of p53 and facilitated HCC cell growth. In summary, we uncovered the pivotal role of NFE2L3 in promoting HCC cell proliferation during proteostasis. This finding may provide a new target for the clinical treatment of HCC.

Small-sample learning reveals propionylation in determining global protein homeostasis

Proteostasis is fundamental for maintaining organismal health. However, the mechanisms underlying its dynamic regulation and how its disruptions lead to diseases are largely unclear. Here, we conduct in-depth propionylomic profiling in Drosophila, and develop a small-sample learning framework to prioritize the propionylation at lysine 17 of H2B (H2BK17pr) to be functionally important. Mutating H2BK17 which eliminates propionylation leads to elevated total protein level in vivo. Further analyses reveal that H2BK17pr modulates the expression of 14.7–16.3% of genes in the proteostasis network, and determines global protein level by regulating the expression of genes involved in the ubiquitin-proteasome system. In addition, H2BK17pr exhibits daily oscillation, mediating the influences of feeding/fasting cycles to drive rhythmic expression of proteasomal genes. Our study not only reveals a role of lysine propionylation in regulating proteostasis, but also implements a generally applicable method which can be extended to other issues with little prior knowledge.

Abstract A56: TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal

Abstract AML with TP53 mutations (TP53Mut) is a poor-risk AML subtype that is largely insensitive to chemotherapy, targeted agents, and hematopoietic stem cell transplantation. TP53Muts are seen in approximately 10-20% of AML and confer a particularly poor prognosis with 1–2-year survival rates to 0-10%. Self-renewal is the defining feature of leukemia stem cells (LSCs) and is central to leukemia progression and relapse. Mutant p53 protein enhances self-renewal in murine models of AML and in human hematopoietic progenitors. In solid tumors, analysis of a variety of TP53Muts defined the proteasome pathway as a common transcriptional target and mediator of oncogenesis among these mutations. Notably, the proteasome degrades I kappa B which activates NF kappa B, which enforces AML self-renewal. Proteasome inhibitors have shown some efficacy in AML clinical trials but have not been well-evaluated in TP53Mut patients. We hypothesize that the proteasome is mediator of self-renewal in TP53Mut AML. To define the transcriptional features of TP53Mut AML, we analyzed the gene expression data in the BEAT AML dataset of primary human AML samples (Tyner, et al. 2018). Relative to TP53WT (n=377 samples), the TP53 mutant samples (n=36) were enriched in every NF kappa B and proteasome gene-set queried. Next, we analyzed a single cell RNA seq dataset of human AML samples (van Galen et al. 2019) and found that the LSC compartment of TP53Mut samples (n=3) strongly upregulated every proteasome gene set we queried (relative to TP53WT samples (n=13)). A recent in vitro screen identified a panel of small molecule inhibitors that reduced viability in TP53-deleted cell lines (Nechiporuk et al. 2019). We tested the activity of these inhibitors in primary human TP53Mut samples. Four of these inhibitors were effective in reducing colony forming capacity or reduced LSC frequency in TP53Mut samples (n=9). We used CyTOF to assess intracellular signaling pathways in LSCs and found that the inhibitors that were most effective in abolishing colony formation and reducing LSC frequency also attenuated NF kappa B levels in TP53Mut LSCs. Accordingly, we found that knockdown of mutant TP53 reduced NF kappa B levels in Kasumi cells (a TP53R248Q AML cell line). Next, we treated a panel of primary human TP53Mut AML samples with the pan-proteasome inhibitors, carfilzomib and bortezomib (FDA approved for lymphoid malignancies) and an immunoproteasome inhibitor, PR957 (in clinical trials for autoimmune conditions). Each drug independently reduced NF kappa B and led to a significant reduction of primary and secondary colony formation. These data suggest that the proteasome/NF kappa B pathway may be an important determinant of self-renewal function in TP53Mut AML and proteasome inhibitors may target the LSCs of this treatment-refractory AML subtype. Citation Format: Marie Lue Antony, Yoonkyu Lee, Klara E Noble-Orcutt, Zohar Sachs. TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A56.

Optimized whole-genome CRISPR interference screens identify ARID1A-dependent growth regulators in human induced pluripotent stem cells.

Perturbing expression is a powerful way to understand the role of individual genes, but can be challenging in important models. CRISPR-Cas screens in human induced pluripotent stem cells (iPSCs) are of limited efficiency due to DNA break-induced stress, while the less stressful silencing with an inactive Cas9 has been considered less effective so far. Here, we developed the dCas9-KRAB-MeCP2 fusion protein for screening in iPSCs from multiple donors. We found silencing in a 200bp window around the transcription start site in polyclonal pools to be as effective as using wild-type Cas9 for identifying essential genes, but with much reduced cell numbers. Whole-genome screens to identify ARID1A-dependent dosage sensitivity revealed the PSMB2 gene, and enrichment of proteasome genes among the hits. This selective dependency was replicated with a proteasome inhibitor, indicating a targetable drug-gene interaction. Many more plausible targets in challenging cell models can be efficiently identified with our approach.

The proteasome regulator Rpn4 controls antifungal drug tolerance by coupling protein homeostasis with metabolic responses to drug stress.

Fungal pathogens overcome antifungal drug therapy by classic resistance mechanisms, such as increased efflux or changes to the drug target. However, even when a fungal strain is susceptible, trailing or persistent microbial growth in the presence of an antifungal drug can contribute to therapeutic failure. This trailing growth is caused by adaptive physiological changes that enable the growth of a subpopulation of fungal cells in high drug concentrations, in what is described as drug tolerance. Mechanistically, antifungal drug tolerance is incompletely understood. Here we report that the transcriptional activator Rpn4 is important for drug tolerance in the human fungal pathogen Candida albicans. Deletion of RPN4 eliminates tolerance to the commonly used antifungal drug fluconazole. We defined the mechanism and show that Rpn4 controls fluconazole tolerance via two target pathways. First, Rpn4 activates proteasome gene expression, which enables sufficient proteasome capacity to overcome fluconazole-induced proteotoxicity and the accumulation of ubiquitinated proteins targeted for degradation. Consistently, inhibition of the proteasome with MG132 eliminates fluconazole tolerance and resistance, and phenocopies the rpn4Δ/Δ mutant for loss of tolerance. Second, Rpn4 is required for wild type expression of the genes required for the synthesis of the membrane lipid ergosterol. Our data indicates that this function of Rpn4 is required for mitigating the inhibition of ergosterol biosynthesis by fluconazole. Based on our findings, we propose that Rpn4 is a central hub for fluconazole tolerance in C. albicans by coupling the regulation of protein homeostasis (proteostasis) and lipid metabolism to overcome drug-induced proteotoxicity and membrane stress.

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.

In-silico identification of novel DDI2 inhibitor in glioblastoma via repurposing FDA approved drugs using molecular docking and MD simulation study

Glioblastoma, the most severe form of brain tumor and a leading cause of death within a year of diagnosis, is characterized by excessive protein synthesis and folding in the lumen of the endoplasmic reticulum (ER), leading to increased ER stress in the cells of GBM tissues. To mitigate this stress the cancer cells have intelligently adopted a plethora of response mechanisms and Unfolded Protein Response (UPR) is one of those. To bear with this exhaustive situation cells upregulate a strong protein degradation system in form of 26S proteasome and blocking of proteasomal gene synthesis may be a potential therapeutic action against GBM. Proteasomal gene synthesis is exclusively dependent on the transcription factor Nuclear respiratory factor 1 (NRF1) and its activating enzyme DNA damage inducible 1 homolog 2 (DDI2). Here in this study, we performed molecular docking against DDI2 with the 20 FDA-approved drugs and identified Alvimopan and Levocabastine as the top two compounds with the best binding score along with the standard drug Nelfinavir. MD simulation (100 ns) of these protein-ligand docked complexes reveals that the stability and compactness of Alvimopan are high in comparison with Nelfinavir. Our in-silico (Molecular docking and Molecular dynamics simulation) studies pointed out that Alvimopan may be repurposed as a DDI2 inhibitor and can be used as a potential anticancer agent for the treatment of brain tumors. Communicated by Ramaswamy H. Sarma

Abstract 1354: Proteasome activation prevents T cell exhaustion and improves antitumor activity

Abstract Tumor infiltrating lymphocytes (TILs) exert their antitumor activity in the tumor microenvironment (TME). Chronic TCR stimulation, together with features of the tumor microenvironment such as hypoxia, result in T cell exhaustion. Exhausted T cells (TEX) are characterized by oxidative stress that leads to cellular accumulation of oxidized proteins. Because the proteasome complex clears oxidized proteins, we hypothesized that its capacity might be overloaded in TEX, limiting their fitness. Analysis of single-cell transcriptomics data from 19 tumor types indicated an increased proteasome activity in TEX compared to non-exhausted TILs. Higher levels of ROS-induced protein oxidation, expression of proteasome genes and proteasome activity were confirmed in human TEX, suggesting that increased proteasome activity might be a compensatory mechanism to exhaustion-associated oxidative stress. We show that pharmacological activation of the proteasome prevented T cell exhaustion, and lead to superior anti-tumor control in the context of adoptive cell therapy in mouse models. Thus, modulation of the proteasome activity is an attractive strategy to prevent T cell dysfunction promoted by the TME, and to potentially overcome this major obstacle in cancer immunotherapies against solid tumors. Citation Format: Ander de Blas, Massimo Andreatta, Asier Antoñana-Vildosola, Paloma Velasco, Leire Egia-Mendikute, Borja Jimenez-Lasheras, Samanta Romina Zanetti, Santiago J. Carmona, Endika Prieto-Fernández, Asis Palazon. Proteasome activation prevents T cell exhaustion and improves antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1354.

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.