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Porcine reproductive and respiratory syndrome (PRRS) is the most economically significant disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Type I interferon (IFN) induces a large number of interferon-stimulated genes (ISGs) expression to inhibit PRRSV infection. To survive in the host, PRRSV has evolved multiple strategies to antagonize host innate immune response. Previous studies have reported that PRRSV N protein decreases the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress IFN-β production. However, whether other PRRSV proteins inhibit the antiviral function of TRIM25 is less well understood. In this study, we first found that PRRSV NSP1α decreased ISGylation of TRIM25. Meanwhile, NSP1α significantly suppressed TRIM25-mediated IFN-β production to promote PRRSV replication. Further studies demonstrated that PRRSV NSP1α reduced the protein level of TRIM25 in proteasome system but did not regulate the transcription level of TRIM25. In addition, the function of NSP1α in TRIM25 degradation did not rely on its papain-like cysteine protease activity. Taken together, PRRSV NSP1α antagonizes the antiviral response of TRIM25 by mediating TRIM25 degradation to promote PRRSV replication. Our data identify TRIM25 as a natural target of PRRSV NSP1α and reveal a novel mechanism that PRRSV induces TRIM25 degradation and inhibits host antiviral immune response., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Basic research to expand treatment options for multiple myeloma is greatly needed due to the refractory nature of the disease. Histone deacetylase (HDAC) inhibitors, which are epigenetic regulators, are attractive but have limited applications. MicroRNAs (miRNAs), which are also epigenetic regulators, are important molecules that may lead to future therapeutic breakthroughs. In this study, we comprehensively searched for miRNAs that are altered by HDAC inhibitors in myeloma cells. We identified miR-7-5p (miR-7) as a miRNA downregulated by HDAC inhibitors. Transfection of myeloma cell lines with miR-7 suppressed cell proliferation, induced apoptosis, and enhanced the effects of the HDAC inhibitor panobinostat. Expression of miR-7 was downregulated by c-Myc inhibition, but upregulated by bortezomib. Comprehensive examination of miR-7 targets revealed four candidates: SLC6A9, LRRC59, EXOSC2, and PSME3. Among these, we focused on PSME3, an oncogene involved in proteasome capacity in myeloma cells. PSME3 knockdown increases myeloma cell death and panobinostat sensitivity. In conclusion, miR-7, which is downregulated by HDAC inhibitors, is a tumor suppressor that targets PSME3. This miR-7 downregulation may be involved in HDAC inhibitor resistance. In addition, combinations of anti-myeloma drugs that complement changes in miRNA expression should be considered., (© 2024. Japanese Society of Hematology.)

The incidence and mortality rates of gastric cancer (GC) remain alarmingly high worldwide, imposing a substantial healthcare burden. In this study, we utilized data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A 4-gene prognostic model was developed to predict patient prognosis, and its accuracy was validated across multiple datasets. Patients with a low-risk score exhibited improved prognosis, elevated tumor mutation burden, heightened sensitivity to both immunotherapy and conventional chemotherapy. Notably, our investigation revealed that the key gene RGS5 positively modulates the expression of mismatch repair proteins via c-Myc. Furthermore, co-immunoprecipitation (COIP) assays demonstrated the interaction between RGS5 and c-Myc. Additionally, we confirmed that RGS5 regulates c-Myc through the ubiquitin-proteasome pathway. Moreover, RGS5 was identified as a positive regulator of PD-L1 expression and exhibited a negative correlation with the majority of immune cells. These findings underscore the potential of RGS5 as a novel biomarker and therapeutic target in the context of GC., (© 2024 Wiley Periodicals LLC.)

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants., (© The Author(s) 2024. Published by Oxford University Press.)

Key Message: Overexpression of rice A20/AN1 zinc-finger protein, OsSAP10, improves water-deficit stress tolerance in Arabidopsis via interaction with multiple proteins. Stress-associated proteins (SAPs) constitute a class of A20/AN1 zinc-finger domain containing proteins and their genes are induced in response to multiple abiotic stresses. The role of certain SAP genes in conferring abiotic stress tolerance is well established, but their mechanism of action is poorly understood. To improve our understanding of SAP gene functions, OsSAP10, a stress-inducible rice gene, was chosen for the functional and molecular characterization. To elucidate its role in water-deficit stress (WDS) response, we aimed to functionally characterize its roles in transgenic Arabidopsis, overexpressing OsSAP10. OsSAP10 transgenics showed improved tolerance to water-deficit stress at seed germination, seedling and mature plant stages. At physiological and biochemical levels, OsSAP10 transgenics exhibited a higher survival rate, increased relative water content, high osmolyte accumulation (proline and soluble sugar), reduced water loss, low ROS production, low MDA content and protected yield loss under WDS relative to wild type (WT). Moreover, transgenics were hypersensitive to ABA treatment with enhanced ABA signaling and stress-responsive genes expression. The protein-protein interaction studies revealed that OsSAP10 interacts with proteins involved in proteasomal pathway, such as OsRAD23, polyubiquitin and with negative and positive regulators of stress signaling, i.e., OsMBP1.2, OsDRIP2, OsSCP and OsAMTR1. The A20 domain was found to be crucial for most interactions but insufficient for all interactions tested. Overall, our investigations suggest that OsSAP10 is an important candidate for improving water-deficit stress tolerance in plants, and positively regulates ABA and WDS signaling via protein-protein interactions and modulation of endogenous genes expression in ABA-dependent manner., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The underlying mechanism of thermotolerance, which is a key virulence factor essential for pathogenic fungi such as Cryptococcus neoformans , is largely unexplored. In this study, our findings suggest that Set302, a homolog of Set3 and a subunit of histone deacetylase complex Set3C, contributes to thermotolerance in C. neoformans . Specifically, the deletion of the predicted Set3C core subunit, Set302, resulted in further reduction in the growth of C. neoformans at 39°C, and survival of transient incubation at 50°C. Transcriptomics analysis revealed that the expression levels of numerous heat stress-responsive genes altered at both 30°C and 39°C due to the lack of Set302. Notably, at 39°C, the absence of Set302 led to the downregulation of gene expression related to the ubiquitin-proteasome system (UPS). Based on the GFP-α-synuclein overexpression model to characterize misfolded proteins, we observed a pronounced accumulation of misfolded GFP-α-synuclein at 39°C, consequently inhibiting C. neoformans thermotolerance. Furthermore, the loss of Set302 exacerbated the accumulation of misfolded GFP-α-synuclein during heat stress. Interestingly, the set302∆ strain exhibited a similar phenotype under proteasome stress as it did at 39°C. Moreover, the absence of Set302 led to reduced production of capsule and melanin. set302∆ strain also displayed significantly reduced pathogenicity and colonization ability compared to the wild-type strain in the murine infection model. Collectively, our findings suggest that Set302 modulates thermotolerance by affecting the degradation of misfolded proteins and multiple virulence factors to mediate the pathogenicity of C. neoformans .IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that poses a potential and significant threat to public health. Thermotolerance plays a crucial role in the wide distribution in natural environments and host colonization of this fungus. Herein, Set302, a critical core subunit for the integrity of histone deacetylase complex Set3C and widely distributed in various fungi and mammals, governs thermotolerance and affects survival at extreme temperatures as well as the formation of capsule and melanin in C. neoformans . Additionally, Set302 participates in regulating the expression of multiple genes associated with the ubiquitin-proteasome system (UPS). By eliminating misfolded proteins under heat stress, Set302 significantly contributes to the thermotolerance of C. neoformans . Moreover, Set302 regulates the pathogenicity and colonization ability of C. neoformans in a murine model. Overall, this study provides new insight into the mechanism of thermotolerance in C. neoformans ., Competing Interests: The authors declare no conflict of interest.

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer)., (© 2024 Gilda et al.)

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The hormone leptin, primarily secreted by adipocytes, plays a crucial role in regulating whole-body energy homeostasis. Homozygous loss-of-function mutations in the leptin gene (LEP) cause hyperphagia and severe obesity, primarily through alterations in leptin's affinity for its receptor or changes in serum leptin concentrations. Although serum concentrations are influenced by various factors (e.g., gene expression, protein synthesis, stability in the serum), proper delivery of leptin from its site of synthesis in the endoplasmic reticulum via the secretory pathway to the extracellular serum is a critical step. However, the regulatory mechanisms and specific machinery involved in this trafficking route, particularly in the context of human LEP mutations, remain largely unexplored. We have employed the Retention Using Selective Hooks system to elucidate the secretory pathway of leptin. We have refined this system into a medium-throughput assay for examining the pathophysiology of a range of obesity-associated LEP variants. Our results reveal that leptin follows the default secretory pathway, with no additional regulatory steps identified prior to secretion. Through screening of leptin variants, we identified three mutations that lead to proteasomal degradation of leptin and one variant that significantly decreased leptin secretion, likely through aberrant disulfide bond formation. These observations have identified novel pathogenic effects of leptin variants, which can be informative for therapeutics and diagnostics. Finally, our novel quantitative screening platform can be adapted for other secreted proteins., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

B7-H4 is an immune checkpoint crucial for inhibiting CD8 + T-cell activity. A clinical trial is underway to investigate B7-H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7-H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7-H4 expression, while EGFR-activating mutants promoted B7-H4 expression through the UPP. We screened B7-H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7-H4 by removing K48- and K63-linked ubiquitin chains from B7-H4, leading to a reduction in B7-H4 degradation. EGFR mutants enhanced B7-H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95 + CD8 + effector T cells and hindered infiltration of Tim-3+CD8 + and LAG-3+CD8 + exhausted T cells by destabilizing B7-H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7-H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7-H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7-H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9 - eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9 - eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

Craniofacial dysmorphisms are among the most common birth defects. Proteasome mutations frequently result in craniofacial dysmorphisms, including lower jaw malformations; however, the underlying mechanisms are unknown. Here, we used a zebrafish proteasome subunit β 1 (psmb1) mutant to define the cellular mechanisms underlying proteasome mutation-induced craniofacial dysmorphisms. psmb1 mutants exhibited a flattened ceratohyal and smaller Meckel's and palatoquadrate cartilages. Ceratohyal flattening was a result of failed chondrocyte convergent extension, accompanied by reduced numbers of chondrocytes in the lower jaw due to defects in chondrocyte differentiation. Morphogenesis of craniofacial muscles and tendons was similarly perturbed. psmb1 mutants lacked the hyohyal muscles, and craniofacial tendons were shortened and disorganized. We additionally identified a critical period for proteasome function in craniofacial development, specifically during chondrocyte and muscle differentiation. psmb1 overexpression in sox10+ cells of mutant embryos rescued both cartilage and tendon phenotypes but induced only a partial rescue of the muscle phenotype, indicating that psmb1 was required in both tissue-autonomous and nonautonomous fashions during craniofacial development. Overall, our work demonstrates that psmb1 is required for craniofacial cartilage, tendon, and muscle differentiation and morphogenesis.

Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells., (© 2024. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yanagi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, we have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. Our findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Our investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health., Competing Interests: Declaration of interests A.C. and K.M. are employees of GeneDx, LLC. J.R.L. has stock in 23andMe and is a paid consultant for Genome International., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

KRAS G12D is the most frequently mutated oncogenic KRAS subtype in solid tumors and remains undruggable in clinical settings. Here, we developed a high affinity, selective, long-acting, and non-covalent KRAS G12D inhibitor, HRS-4642, with an affinity constant of 0.083 nM. HRS-4642 demonstrated robust efficacy against KRAS G12D-mutant cancers both in vitro and in vivo. Importantly, in a phase 1 clinical trial, HRS-4642 exhibited promising anti-tumor activity in the escalating dosing cohorts. Furthermore, the sensitization and resistance spectrum for HRS-4642 was deciphered through genome-wide CRISPR-Cas9 screening, which unveiled proteasome as a sensitization target. We further observed that the proteasome inhibitor, carfilzomib, improved the anti-tumor efficacy of HRS-4642. Additionally, HRS-4642, either as a single agent or in combination with carfilzomib, reshaped the tumor microenvironment toward an immune-permissive one. In summary, this study provides potential therapies for patients with KRAS G12D-mutant cancers, for whom effective treatments are currently lacking., Competing Interests: Declaration of interests C.Z. reported honoraria as a speaker from Roche, Lily China, Boehringer Ingelheim, Merck, Hengrui, Qilu, Sanofi, Merck Sharp & Dohme, Innovent Biologics, C-Stone, Luye Pharma, TopAlliance Biosciences, and Amoy Diagnostics and advisor fees for Innovent Biologics, Hengrui, Qilu, and TopAlliance Biosciences. We also declare a patent application (CN117462688A) filed by Jiangsu Hengrui Pharmaceuticals Co., Ltd., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is a globally prevalent cancer with significant mortality rates. OSCC a predominant subtype of head and neck squamous cell carcinoma (HNSCC), poses a substantial health burden. Despite advancements in diagnosis and therapy, OSCC prognosis remains poor. The 26S proteasome, a cellular protein degradation complex, is associated with cancer, including PSMA1, a proteasomal subunit, which is upregulated in various cancers. We analyzed PSMA1 expression using TCGA data, validated it in OSCC samples using real-time PCR, and explored its role through various databases. Tumor and adjacent normal tissues from OSCC patients were examined, and PSMA1 expression was analyzed. Survival analysis assessed the impact of PSMA1 on patient outcomes, while immune infiltration was examined using the TIMER database. GeneMANIA, STRING, and Metascape were utilized for gene interaction and pathway analysis. PSMA1 was significantly upregulated in OSCC and HNSCC. Its overexpression correlated with advanced clinicopathological features and poorer prognosis in HNSCC patients. PSMA1 expression is also related to immune cell infiltration. Gene interaction analysis revealed PSMA1 involvement in proteolysis regulation, suggesting its potential as a therapeutic target. PSMA1 upregulation in HNSCC association with adverse clinicopathological features and prognosis underscores its potential significance. Further research is warranted to elucidate its molecular mechanisms and therapeutic potential in OSCC management., (© 2024. The Author(s), under exclusive licence to The Society of The Nippon Dental University.)

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, characterized by high morbidity and mortality rates, as well as unfavorable treatment outcomes. Tripartite motif-containing protein 47 (TRIM47) has been implicated in various diseases including tumor progression with the activity of E3 ubiquitin ligase. However, the precise regulatory mechanisms underlying the involvement of TRIM47 in HCC remain largely unexplored. Here, we provide evidence that TRIM47 exhibits heightened expression in tumor tissues, and its expression is in intimate association with clinical staging and patient prognosis. TRIM47 promotes HCC proliferation, migration, and invasion as an oncogene by in vitro gain- and loss-of-function experiments. TRIM47 knockdown results in HCC ferroptosis induction, primarily through CDO1 involvement to regulate GSH synthesis. Subsequent experiments confirm the interaction between TRIM47 and CDO1 dependent on B30.2 domain, wherein TRIM47 facilitates K48-linked ubiquitination, leading to a decrease in CDO1 protein abundance in HCC. Furthermore, CDO1 is able to counteract the promotional effect of TRIM47 on HCC biological functions. Overall, our research provides novel insight into the mechanism of TRIM47 in CDO1-mediated ferroptosis in HCC cells, highlighting its value as a potential target candidate for HCC therapeutic approaches., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification and functional characterization in teleosts are still rarely known. In this work, we cloned the otub1 of miiuy croaker (Miichthys miiuy), analyzed its sequence, structure, and evolution at genetic and protein levels, and determined its function in the antiviral immune response. The complete open reading frame (ORF) of miiuy croaker otub1 is 843 bp in length, encoding 280 amino acids. Miiuy croaker Otub1 has an OTU domain at the carboxyl terminus, which is a common functional domain that exists in OTU deubiquitinases. Molecular characteristics and evolution analysis results indicated that miiuy croaker Otub1, especially its functional domain, is highly conserved during evolution. The luciferase reporter assays showed that miiuy croaker Otub1 could significantly inhibit the poly(I:C) and Irf3-induced IFN1 and IFN-stimulated response element (ISRE) activation. Further experiments showed that miiuy croaker Otub1 decreases Irf3 protein abundance by promoting its proteasomal degradation. These data suggest that the evolutionarily conserved Otub1 acts as a suppressor in controlling antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: MG132, a proteasome inhibitor, is widely used to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity by proteasome-mediated degradation of IκB. It has been marketed as a specific, reversible, cell-permeable and low-cost inhibitor. However, adverse effects of the compound have been reported in the literature. We recently discovered and characterised a point mutation in the acute phase protein serum amyloid A (SAA) in chickens, by overexpressing the protein in chicken hepatocellular carcinoma (LMH) cells. This serine to arginine exchange at amino acid position 90 (SAA.R90S) leads to intra- and extracellular accumulation of SAA, which is surprisingly counteracted by MG132 treatment, independent of SAA's intrinsic promoter., Methods and Results: To test, whether low proteasomal degradation of SAA.R90S is responsible for the observed intra- and extracellular SAA accumulation, we intended to inhibit the proteasome in SAA wild type (SAA.WT) overexpressing cells with MG132. However, we observed an unexpected drastic decrease in SAA protein expression at the transcript level. NF-κB gene expression was unchanged by MG132 at the measured time point., Conclusions: The observed results demonstrate that MG132 inhibits SAA expression at the transcript level, independent of its endogenous promoter. Further, the data might indicate that NF-κB is not involved in the observed MG132-induced inhibition of SAA expression. We, consequently, question in this brief report whether MG132 should truly be categorised as a specific ubiquitin proteasome inhibitor and recommend the usage of alternative compounds., (© 2024. The Author(s).)

We conducted transcriptome sequencing on salt-tolerant mutants X5 and X3, and a control (Ctr) strain of Gracilariopsis lemaneiformis after treatment with artificial seawater at varying salinities (30‱, 45‱, and 60‱) for 3 weeks. Differentially expressed genes were identified and a weighted co-expression network analysis was conducted. The blue, red, and tan modules were most closely associated with salinity, while the black, cyan, light cyan, and yellow modules showed a close correlation with strain attributes. KEGG enrichment of genes from the aforementioned modules revealed that the key enrichment pathways for salinity attributes included the proteasome and carbon fixation in photosynthesis, whereas the key pathways for strain attributes consisted of lipid metabolism, oxidative phosphorylation, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) interactions in vesicular transport, and porphyrin and chlorophyll metabolism. Gene expression for the proteasome and carbon fixation in photosynthesis was higher in all strains at 60‱. In addition, gene expression in the proteasome pathway was higher in the X5-60 than Ctr-60 and X3-60. Based on the above data and relevant literature, we speculated that mutant X5 likely copes with high salt stress by upregulating genes related to lysosome and carbon fixation in photosynthesis. The proteasome may be reset to adjust the organism's proteome composition to adapt to high-salt environments, while carbon fixation may aid in maintaining material and energy metabolism for normal life activities by enhancing carbon dioxide uptake via photosynthesis. The differences between the X5-30 and Ctr-30 expression of genes involved in the synthesis of secondary metabolites, oxidative phosphorylation, and SNARE interactions in vesicular transport suggested that the X5-30 may differ from Ctr-30 in lipid metabolism, energy metabolism, and vesicular transport. Finally, among the key pathways with good correlation with salinity and strain traits, the key genes with significant correlation with salinity and strain traits were identified by correlation analysis.

Human and simian immunodeficiency viruses (HIV and SIV) are lentiviruses that reverse transcribe their RNA genome with subsequent integration into the genome of the target cell. How progressive infection and administration of antiretrovirals (ARVs) longitudinally influence the transcriptomic and epigenetic landscape of particular T cell subsets, and how these may influence the genetic location of integration are unclear. Here, we use RNAseq and ATACseq to study the transcriptomics and epigenetic landscape of longitudinally sampled naïve and memory CD4+ and CD8+ T cells in two species of non-human primates prior to SIV infection, during chronic SIV infection, and after administration of ARVs. We find that SIV infection leads to significant alteration to the transcriptomic profile of all T cell subsets that are only partially reversed by administration of ARVs. Epigenetic changes were more apparent in animals with longer periods of untreated SIV infection and correlated well with changes in corresponding gene expression. Known SIV integration sites did not vary due to SIV status but did contain more open chromatin in rhesus macaque memory T cells, and the expression of proteasome-related genes at the pre-SIV timepoint correlated with subsequent viremia.IMPORTANCEChronic inflammation during progressive human and simian immunodeficiency virus (HIV and SIV) infections leads to significant co-morbidities in infected individuals with significant consequences. Antiretroviral (ARV)-treated individuals also manifest increased levels of inflammation which are associated with increased mortalities. These data will help guide rational development of modalities to reduce inflammation observed in people living with HIV and suggest mechanisms underlying lentiviral integration site preferences., Competing Interests: The authors declare no conflict of interest.

Proteasome 26S Subunit, Non-ATPase 9 ( psmd9 ) plays an important role in the balance of protamine and the stability of the nucleolar structure during spermatogenesis. In this study, we cloned the psmd9 of Cynoglossus semilaevis and analyzed its expression pattern. psmd9 was identified on the Z chromosome of C. semilaevis , which is considered an interesting candidate gene for spermatogenesis. qRT-PCR and FISH experiments showed that the psmd9 gene was significantly highly expressed in the testes. It is worth noting that the expression level of psmd9 in male fish testes is significantly higher than that in pseudomales. In order to further explore the role of psmd9 in spermatogenesis, a male testicular cell line was used as the experimental material. The results of the psmd9 -RNAi and overexpression experiments showed that psmd9 had a synergistic effect with spermatogenesis-related genes dnd1 , cfap69 , dnah3 and dnajb13 , but had an antagonistic effect with ccne2 . Our findings offer a scientific foundation for comprehending the role of psmd9 in the spermatogenesis regulatory network of C. semilaevis .

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

PGM1 -linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.

Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress., (© 2024 Ward et al.)

Around 10% of Parkinson's disease (PD) cases are associated with mutations in various genes, including FBXO7, which encodes the substrate-recognition component for the Skp1-Cullin-F-box (SCF) class of ubiquitin E3 ligases that target proteins for proteasomal degradation. In their recent study, Al Rawi et al. characterized a new mutation in FBXO7, L250P, in a pediatric patient. Their findings reveal that the L250P mutation abolishes Fbxo7 interaction with the proteasome regulator, proteasome inhibitor 31kD (PI31), affecting proteasomal activity and the ubiquitination of some of the ligase's targets. Furthermore, the authors show that this previously undescribed mutation impairs mitochondrial function and mitophagy, emphasizing the importance of mitochondrial and proteasomal dysfunction in PD pathogenesis., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy., Methods: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells., Results: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities., Conclusion: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 SCF Fbxo7 -mediated ubiquitination of MiD49. The L250P mutation reduces the SCF Fbxo7 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 SCF Fbxo7 E3 ubiquitin ligase activity., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy-an imbalance in chromosome copy numbers-is frequent 1,2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory 3-5 . Here we generate a proteomic resource and merge it with genomic 1 and transcriptomic 6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins-especially subunits of protein complexes-show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes., (© 2024. The Author(s).)

Recent genome-wide association studies have identified a missense variant p.A165T in mitochondrial amidoxime-reducing component 1 (mARC1) that is strongly associated with protection from all-cause cirrhosis and improved prognosis in nonalcoholic steatohepatitis. The precise mechanism of this protective effect is unknown. Substitution of alanine 165 with threonine is predicted to affect mARC1 protein stability and to have deleterious effects on its function. To investigate the mechanism, we have generated a knock-in mutant mARC1 A165T and a catalytically dead mutant C273A (as a control) in human hepatoma HepG2 cells, enabling characterization of protein subcellular distribution, stability, and biochemical functions of the mARC1 mutant protein expressed from its endogenous locus. Compared to WT mARC1, we found that the A165T mutant exhibits significant mislocalization outside of its traditional location anchored in the mitochondrial outer membrane and reduces protein stability, resulting in lower basal levels. We evaluated the involvement of the ubiquitin proteasome system in mARC1 A165T degradation and observed increased ubiquitination and faster degradation of the A165T variant. In addition, we have shown that HepG2 cells carrying the MTARC1 p.A165T variant exhibit lower N-reductive activity on exogenously added amidoxime substrates in vitro. The data from these biochemical and functional assays suggest a mechanism by which the MTARC1 p.A165T variant abrogates enzyme function which may contribute to its protective effect in liver disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Radiation-induced enteritis, a significant concern in abdominal radiation therapy, is associated closely with gut microbiota dysbiosis. The mucus layer plays a pivotal role in preventing the translocation of commensal and pathogenic microbes. Although significant expression of REGγ in intestinal epithelial cells is well established, its role in modulating the mucus layer and gut microbiota remains unknown. The current study revealed notable changes in gut microorganisms and metabolites in irradiated mice lacking REGγ, as compared to wild-type mice. Concomitant with gut microbiota dysbiosis, REGγ deficiency facilitated the infiltration of neutrophils and macrophages, thereby exacerbating intestinal inflammation after irradiation. Furthermore, fluorescence in situ hybridization assays unveiled an augmented proximity of bacteria to intestinal epithelial cells in REGγ knockout mice after irradiation. Mechanistically, deficiency of REGγ led to diminished goblet cell populations and reduced expression of key goblet cell markers, Muc2 and Tff3, observed in both murine models, minigut organoid systems and human intestinal goblet cells, indicating the intrinsic role of REGγ within goblet cells. Interestingly, although administration of broad-spectrum antibiotics did not alter the goblet cell numbers or mucin 2 (MUC2) secretion, it effectively attenuated inflammation levels in the ileum of irradiated REGγ absent mice, bringing them down to the wild-type levels. Collectively, these findings highlight the contribution of REGγ in counteracting radiation-triggered microbial imbalances and cell-autonomous regulation of mucin secretion., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

APE2 plays important roles in the maintenance of genomic and epigenomic stability including DNA repair and DNA damage response. Accumulating evidence has suggested that APE2 is upregulated in multiple cancers at the protein and mRNA levels and that APE2 upregulation is correlative with higher and lower overall survival of cancer patients depending on tumor type. However, it remains unknown how APE2 protein abundance is maintained and regulated in cells. Here, we provide the first evidence of APE2 regulation via the posttranslational modification ubiquitin. APE2 is poly-ubiquitinated via K48-linked chains and degraded via the ubiquitin-proteasome system where K371 is the key residue within APE2 responsible for its ubiquitination and degradation. We further characterize MKRN3 as the E3 ubiquitin ligase for APE2 ubiquitination in cells and in vitro. In summary, this study offers the first definition of the APE2 proteostasis network and lays the foundation for future studies pertaining to the posttranslational modification regulation and functions of APE2 in genome integrity and cancer etiology/treatment., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The ubiquitin-proteasome system (UPS) is an essential regulatory system for maintaining homeostasis, and its dysfunction may cause various diseases. The activity of proteasome and ubiquitin-conjugating enzymes has been found to be greatly increased in breast cancer (BC), indicating that the heterogeneity of UPS may be related to the progression of BC. Gene data was obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases and performed in multiple algorithms to construct a UPS-related signature for BC. Patients in the UPS low-risk group had greater overall and recurrence-free survival probability than those in the UPS high-risk group. This signature was closely associated with functional enrichment. Some high metabolism-related pathways were more active in the UPS high-risk group. The UPS low-risk group had more abundant anti-tumor immune cells, while in the UPS high-risk group, immunosuppressive cells were dominant. More importantly, we found that the UPS low-risk group was more sensitive to immunotherapy, while the UPS high-risk group responded better to radiotherapy. Drug sensitivity analysis identified more effective chemotherapy drugs in different UPS-related risk groups. This UPS-related signature may serve as a novel biomarker and independent prognostic factor for BC. It can effectively predict prognosis, immune infiltration, and therapy efficacy, providing new strategies for individualized treatment., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Our and other's laboratory microarray-derived transcriptomic studies in human PD substantia nigra pars compacta (SNpc) samples have opened an avenue to concentrate on potential gene intersections or cross-talks along the dopaminergic (DAergic) neurodegenerative cascade in sporadic PD (SPD). One emerging gene candidate identified was SKP1A (p19, S-phase kinase-associated protein 1A), found significantly decreased in the SNpc as confirmed later at the protein level. SKP1 is part of the Skp1, Cullin 1, F-box protein (SCF) complex, the largest known class of sophisticated ubiquitin-proteasome/E3-ligases and was found to directly interact with FBXO7, a gene defective in PARK15-linked PD. This finding has led us to the hypothesis that a targeted site-specific reduction of Skp1 levels in DAergic neuronal cell culture and animal systems may result in a progressive loss of DAergic neurons and hopefully recreate motor disabilities in animals. The second premise considers the possibility that both intrinsic and extrinsic factors (e.g., manipulation of selected genes and mitochondria impairing toxins), alleged to play central roles in DAergic neurodegeneration in PD, may act in concert as modifiers of Skp1 deficiency-induced phenotype alterations ('dual-hit' hypothesis of neurodegeneration). To examine a possible role of Skp1 in DAergic phenotype, we have initially knocked down the expression of SKP1A gene in an embryonic mouse SN-derived cell line (SN4741) with short hairpin RNA (shRNA) lentiviruses (LVs). The deficiency of SKP1A closely recapitulated cardinal features of the DAergic pathology of human PD, such as decreased expression of DAergic phenotypic markers and cell cycle aberrations. Furthermore, the knocked down cells displayed a lethal phenotype when induced to differentiate exhibiting proteinaceous round inclusion structures, which were almost identical in composition to human Lewy bodies, a hallmark of PD. These findings support a role for Skp1 in neuronal phenotype, survival, and differentiation. The identification of Skp1 as a key player in DAergic neuron function suggested that a targeted site-specific reduction of Skp1 levels in mice SNpc may result in a progressive loss of DAergic neurons and terminal projections in the striatum. The injected LV SKP1shRNA to mouse SN resulted in decreased expression of Skp1 protein levels within DAergic neurons and loss of tyrosine hydroxylase immunoreactivity (TH-IR) in both SNpc and striatum that was accompanied by time-dependent motor disabilities. The reduction of the vertical movements, that is rearing, may be reminiscent of the early occurrence of hypokinesia and axial, postural instability in PD. According to the 'dual-hit' hypothesis of neurodegenerative diseases, it is predicted that gene-gene and/or gene-environmental factors would act in concert or sequentially to propagate the pathological process of PD. Our findings are compatible with this conjecture showing that the genetic vulnerability caused by knock down of SKP1A renders DAergic SN4741 cells especially sensitive to genetic reduction of Aldh1 and exposure to the external stressors MPP + and DA, which have been implicated in PD pathology. Future consideration should be given in manipulation SKP1A expression as therapeutic window, via its induction genetically or pharmacological, to prevent degeneration of the nigra striatal dopamine neurons, since UPS is defective., (© 2023. The Author(s).)

The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations in the TP53 gene, which not only abrogate the tumor-suppressive functions but also confer p53 mutant proteins with oncogenic potential. The latter is achieved through so-called gain-of-function (GOF) mutations that promote cancer progression, metastasis, and therapy resistance by deregulating transcriptional networks, signaling pathways, metabolism, immune surveillance, and cellular compositions of the microenvironment. Despite recent progress in understanding the complexity of mutp53 in neoplastic development, the exact mechanisms of how mutp53 contributes to cancer development and how they escape proteasomal and lysosomal degradation remain only partially understood. In this review, we address recent findings in the field of oncogenic functions of mutp53 specifically regarding, but not limited to, its implications in metabolic pathways, the secretome of cancer cells, the cancer microenvironment, and the regulating scenarios of the aberrant proteasomal degradation. By analyzing proteasomal and lysosomal protein degradation, as well as its connection with autophagy, we propose new therapeutical approaches that aim to destabilize mutp53 proteins and deactivate its oncogenic functions, thereby providing a fundamental basis for further investigation and rational treatment approaches for TP53-mutated cancers.

Bladder cancer (BC) is a malignant tumor of the urinary system with high mortality and recurrence rates. Proteasome subunit type 4 (PSMB4) is highly expressed and has been identified as having oncogenic properties in a variety of cancer types. This study aimed to explore the effect of PSMB4 knockdown on the survival, migration, and angiogenesis of human bladder cancer cells with different degrees of malignancy. We analyzed the effects of PSMB4 knockdown in bladder cancer cells and endothelial cells in the tumor microenvironment. PSMB4 was highly expressed in patients with low- and high-grade urothelial carcinoma. Inhibition of PSMB4 reduced protein expression of focal adhesion kinase (FAK) and myosin light chain (MLC), leading to reduced migration. Furthermore, the suppression of PSMB4 decreased the levels of vascular endothelial factor B (VEGF-B), resulting in lower angiogenic abilities in human bladder cancer cells. PSMB4 inhibition affected the migratory ability of HUVECs and reduced VEGFR2 expression, consequently downregulating angiogenesis. In the metastatic animal model, PSMB4 knockdown reduced the relative volumes of lung tumors. Our findings suggest the role of PSMB4 as a potential target for therapeutic strategies against human bladder cancer.

Background: Mounting evidences shows that the ubiquitin‒proteasome pathway plays a pivotal role in tumor progression. The expression of 26S proteasome non-ATPase regulatory subunit 9 (PSMD9) is correlated with recurrence and radiotherapy resistance in several tumor types. However, the role and mechanism of PSMD9 in hepatocellular carcinoma (HCC) progression remain largely unclear., Methods: PSMD9 was identified as a prognosis-related biomarker for HCC based on analysis of clinical characteristics and RNA-seq data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and the JP Project of the International Cancer Genome Consortium (ICGC-LIRI-JP). PSMD9 expression was analyzed in cancer tissues and adjacent noncancerous tissues via immunohistochemistry and Western blotting. Multiple in vivo and in vitro experimental techniques (such as CCK-8, colony formation, EdU, and Transwell assays; flow cytometry; Western blotting; quantitative RT-PCR; Coimmunoprecipitation assay and immunofluorescence confocal imaging) were used to assess the functions of PSMD9 in the pathogenesis of HCC., Results: We found that the expression of PSMD9 was upregulated and associated with a poor prognosis in HCC patients. PSMD9 promoted HCC cell proliferation, migration, invasion and metastasis. Knockdown of PSMD9 significantly inhibited HCC cell proliferation by inducing G1/S cell cycle arrest and apoptosis. Mechanistically, we demonstrated that PSMD9 promoted HCC cell proliferation and metastasis via direct interaction with the E3 ubiquitin ligase c-Cbl, suppresses EGFR ubiquitination, influenced EGFR endosomal trafficking and degradation and subsequently activated ERK1/2 and Akt signaling. In addition, we showed that PSMD9 knockdown sensitized HCC cells to the tyrosine kinase inhibitor erlotinib in vitro and in vivo., Conclusions: Collectively, our results indicate that PSMD9 drives HCC progression and erlotinib resistance by suppressing c-Cbl mediated EGFR ubiquitination and therefore can be a potential therapeutic target for HCC., (© 2024. The Author(s).)

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness., (© 2024. The Author(s).)

Neuromuscular disorders caused by dysfunction of the mitochondrial respiratory chain are common, severe and untreatable. We recovered a number of mitochondrial genes, including electron transport chain components, in a large forward genetic screen for mutations causing age-related neurodegeneration in the context of proteostasis dysfunction. We created a model of complex I deficiency in the Drosophila retina to probe the role of protein degradation abnormalities in mitochondrial encephalomyopathies. Using our genetic model, we found that complex I deficiency regulates both the ubiquitin/proteasome and autophagy/lysosome arms of the proteostasis machinery. We further performed an in vivo kinome screen to uncover new and potentially druggable mechanisms contributing to complex I related neurodegeneration and proteostasis failure. Reduction of RIOK kinases and the innate immune signaling kinase pelle prevented neurodegeneration in complex I deficiency animals. Genetically targeting oxidative stress, but not RIOK1 or pelle knockdown, normalized proteostasis markers. Our findings outline distinct pathways controlling neurodegeneration and protein degradation in complex I deficiency and introduce an experimentally facile model in which to study these debilitating and currently treatment-refractory disorders., (© The Author(s) 2024. Published by Oxford University Press.)

Most mesenchymal tumors found in the uterine corpus are benign tumors; however, uterine leiomyosarcoma is a malignant tumor with unknown risk factors that repeatedly recurs and metastasizes. In some cases, the histopathologic findings of uterine leiomyoma and uterine leiomyosarcoma are similar and surgical pathological diagnosis using excised tissue samples is difficult. It is necessary to analyze the risk factors for human uterine leiomyosarcoma and establish diagnostic biomarkers and treatments. Female mice deficient in the proteasome subunit low molecular mass peptide 2 (LMP2)/β1i develop uterine leiomyosarcoma spontaneously., Material and Methods: Out of 334 patients with suspected uterine mesenchymal tumors, patients diagnosed with smooth muscle tumors of the uterus were selected from the pathological file. To investigate the expression status of biomarker candidate factors, immunohistochemical staining was performed with antibodies of biomarker candidate factors on thin-cut slides of human uterine leiomyosarcoma, uterine leiomyoma, and other uterine mesenchymal tumors., Results and Discussion: In human uterine leiomyosarcoma, there was a loss of LMP2/β1i expression and enhanced cyclin E1 and Ki-67/MIB1 expression. In human uterine leiomyomas and normal uterine smooth muscle layers, enhanced LMP2/β1i expression and the disappearance of the expression of E1 and Ki-67/MIB1 were noted. The pattern of expression of each factor in other uterine mesenchymal tumors was different from that of uterine leiomyosarcoma., Conclusions: LMP2/β1i, cyclin E1, and Ki-67/MIB1 may be candidate factors for biomarkers of human uterine leiomyosarcoma. Further large-cohort clinical trials should be conducted to establish treatments and diagnostics for uterine mesenchymal tumors.

Group A Rotavirus (RVA) is a major cause of diarrhea in infants and piglets. β2-microglobulin (β2 M), encoded by the B2M gene, serves as a crucial subunit of the major histocompatibility complex class I (MHC-I) molecules. β2 M is indispensable for the transport of MHC-I to the cell membrane. MHC-I, also known as swine leukocyte antigen class I (SLA-I) in pigs, presents viral antigens to the cell surface. In this study, RVA infection down-regulated β2 M expression in both porcine intestinal epithelial cells-J2 (IPEC-J2) and MA-104 cells. RVA infection did not down-regulate the mRNA level of the B2M gene, indicating that the down-regulation of β2 M occurred on the protein level. Mechanismly, RVA infection triggered β2 M aggregation in the endoplasmic reticulum (ER) and enhanced the Lys48 (K48)-linked ubiquitination of β2 M, leading to the degradation of β2 M through ERAD-proteasome pathway. Furthermore, we found that RVA infection significantly impeded the level of SLA-I on the surface, and the overexpression of β2 M could recover its expression. In this study, our study demonstrated that RVA infection degrades β2 M via ERAD-proteasome pathway, consequently hampering SLA-I expression on the cell surface. This study would enhance the understanding of the mechanism of how RVA infection induces immune escape., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DNA-protein crosslinks (DPCs) induced by aldehydes interfere with replication and transcription. Hereditary deficiencies in DPC repair and aldehyde clearance processes cause progeria, including Ruijs-Aalfs syndrome (RJALS) and AMeD syndrome (AMeDS) in humans. Although the elimination of DPC during replication has been well established, how cells overcome DPC lesions in transcription remains elusive. Here we show that endogenous aldehyde-induced DPC roadblocks are efficiently resolved by transcription-coupled repair (TCR). We develop a high-throughput sequencing technique to measure the genome-wide distribution of DPCs (DPC-seq). Using proteomics and DPC-seq, we demonstrate that the conventional TCR complex as well as VCP/p97 and the proteasome are required for the removal of formaldehyde-induced DPCs. TFIIS-dependent cleavage of RNAPII transcripts protects against transcription obstacles. Finally, a mouse model lacking both aldehyde clearance and TCR confirms endogenous DPC accumulation in actively transcribed regions. Collectively, our data provide evidence that transcription-coupled DPC repair (TC-DPCR) as well as aldehyde clearance are crucial for protecting against metabolic genotoxin, thus explaining the molecular pathogenesis of AMeDS and other disorders associated with defects in TCR, such as Cockayne syndrome., (© 2024. The Author(s).)

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) ( p  < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Plant inflorescence architecture is determined by inflorescence meristem (IM) activity and controlled by genetic mechanisms associated with environmental factors. In Arabidopsis (Arabidopsis thaliana), TERMINAL FLOWER1 (TFL1) is expressed in the IM and is required to maintain indeterminate growth, whereas LEAFY (LFY) is expressed in the floral meristems (FMs) formed at the periphery of the IM and is required to activate determinate floral development. Here, we address how Arabidopsis indeterminate inflorescence growth is determined. We show that the 26S proteasome subunit REGULATORY PARTICLE AAA-ATPASE 2a (RPT2a) is required to maintain the indeterminate inflorescence architecture in Arabidopsis. rpt2a mutants display reduced TFL1 expression levels and ectopic LFY expression in the IM and develop a determinate zigzag-shaped inflorescence. We further found that RPT2a promotes DNA METHYLTRANSFERASE1 degradation, leading to DNA hypomethylation upstream of TFL1 and high TFL1 expression levels in the wild-type IM. Overall, our work reveals that proteolytic input into the epigenetic regulation of TFL1 expression directs inflorescence architecture in Arabidopsis, adding an additional layer to stem cell regulation., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The 26S proteasome is the major protein degradation machinery in cells. Cancer cells use the proteasome to modulate gene expression networks that promote tumor growth. Proteasome inhibitors have emerged as effective cancer therapeutics, but how they work mechanistically remains unclear. Here, using integrative genomic analysis, we discovered unexpected reprogramming of the chromatin landscape and RNA polymerase II (RNAPII) transcription initiation in breast cancer cells treated with the proteasome inhibitor MG132. The cells acquired dynamic changes in chromatin accessibility at specific genomic loci termed differentially open chromatin regions (DOCR). DOCRs with decreased accessibility were promoter proximal and exhibited unique chromatin architecture associated with divergent RNAPII transcription. Conversely, DOCRs with increased accessibility were primarily distal to transcription start sites and enriched in oncogenic superenhancers predominantly accessible in non-basal breast tumor subtypes. These findings describe the mechanisms by which the proteasome modulates the expression of gene networks intrinsic to breast cancer biology., Significance: Our study provides a strong basis for understanding the mechanisms by which proteasome inhibitors exert anticancer effects. We find open chromatin regions that change during proteasome inhibition, are typically accessible in non-basal breast cancers., (© 2024 The Authors; Published by the American Association for Cancer Research.)

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 14 years, 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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mutations in proteasome β-subunits or their chaperone and regulatory proteins are associated with proteasome-associated autoinflammatory disorders (PRAAS). We studied six unrelated infants with three de novo heterozygous missense variants in PSMB10, encoding the proteasome β2i-subunit. Individuals presented with T-B-NK± severe combined immunodeficiency (SCID) and clinical features suggestive of Omenn syndrome, including diarrhea, alopecia, and desquamating erythematous rash. Remaining T cells had limited T cell receptor repertoires, a skewed memory phenotype, and an elevated CD4/CD8 ratio. Bone marrow examination indicated severely impaired B cell maturation with limited V(D)J recombination. All infants received an allogeneic stem cell transplant and exhibited a variety of severe inflammatory complications thereafter, with 2 peri-transplant and 2 delayed deaths. The single long-term transplant survivor showed evidence for genetic rescue through revertant mosaicism overlapping the affected PSMB10 locus. The identified variants (c.166G>C [p.Asp56His] and c.601G>A/c.601G>C [p.Gly201Arg]) were predicted in silico to profoundly disrupt 20S immunoproteasome structure through impaired β-ring/β-ring interaction. Our identification of PSMB10 mutations as a cause of SCID-Omenn syndrome reinforces the connection between PRAAS-related diseases and SCID., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)