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

The ubiquitin-proteasome system (UPS) has a critical role in post-translational protein modification that is essential for the maintenance of all cellular functions, including immune responses. The proteasome complex is ubiquitously expressed and is responsible for degradation of short-lived structurally abnormal, misfolded and not-needed proteins that are targeted for degradation via ubiquitin conjugation. Over the last 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.

Proteasome Inhibition Reprograms Chromatin Landscape in Breast Cancer.

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.

Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy.

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.

The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.

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

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

Rotavirus infection inhibits SLA-I expression on the cell surface by degrading ß2 M via ERAD-proteasome pathway.

Group A Rotavirus (RVA) is a major cause of diarrhea in infants and piglets. ß2-microglobulin (ß2 M), encoded by the B2M gene, serves as a crucial subunit of the major histocompatibility complex class I (MHC-I) molecules. ß2 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 ß2 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 ß2 M occurred on the protein level. Mechanismly, RVA infection triggered ß2 M aggregation in the endoplasmic reticulum (ER) and enhanced the Lys48 (K48)-linked ubiquitination of ß2 M, leading to the degradation of ß2 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 ß2 M could recover its expression. In this study, our study demonstrated that RVA infection degrades ß2 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.

Expanding the PRAAS spectrum: De novo mutations of immunoproteasome subunit ß-type 10 in six infants with SCID-Omenn syndrome.

Mutations in proteasome ß-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 ß2i-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 ß-ring/ß-ring interaction. Our identification of PSMB10 mutations as a cause of SCID-Omenn syndrome reinforces the connection between PRAAS-related diseases and SCID.

PSMB2 plays an oncogenic role in glioma and correlates to the immune microenvironment.

There has been an upward trend in the incidence of glioma, with high recurrence and high mortality. The beta subunits of the 20S proteasome are encoded by the proteasome beta (PSMB) genes and may affect the proteasome's function in glioma, assembly and inhibitor binding. This study attempted to reveal the function of the proliferation and invasion of glioma cells, which is affected by proteasome 20S subunit beta 2 (PSMB2). We subjected the data downloaded from the TCGA database to ROC, survival, and enrichment analyses. After establishing the stable PSMB2 knockdown glioma cell line. We detect the changes in the proliferation, invasion and migration of glioma cells by plate colony formation assay, transwell assay, wound healing assay and flow cytometry and PSMB2 expression was verified by quantitative PCR and Western blotting to identify the mRNA and protein levels. PSMB2 expression was higher in glioma tissues, and its expression positively correlated with poor prognosis and high tumor grade and after PSMB2 knockdown, the proliferation, invasion and migration of glioma cells were weakened.

Mutation in the 26S proteasome regulatory subunit rpn2 gene in Plasmodium falciparum confers resistance to artemisinin.

Introduction: Malaria parasites increasingly develop resistance to all drugs available in the market, hampering the goal of reducing malaria burden. Methods: Herein, we evaluated the impact of a single-nucleotide variant, E738K, present in the 26S proteasome regulatory subunit rpn2 gene, identified in Plasmodium chabaudi resistant parasites. Plasmids carrying a functional rpn2 interspecies chimeric gene with 5' recombination region from P. falciparum and 3' from P. chabaudi were constructed and transfected into Dd2 P. falciparum parasites. Results and discussion: The 738K variant parasite line presented increased parasite survival when subjected to dihydroartemisinin (DHA), as well as increased chymotrypsin-like activity and decreased accumulation of polyubiquitinated proteins. We thus conclude that the ubiquitin-proteasome pathway, including the 738K variant, play an important role in parasite response to DHA, being the first report of a mutation in a potential DHA drug target enhancing parasite survival and contributing to a significant advance in the understanding the biology of artemisinin resistance.

Sex- and region-specific cortical and hippocampal whole genome transcriptome profiles from control and APP/PS1 Alzheimer's disease mice.

A variety of Alzheimer's disease (AD) mouse models has been established and characterized within the last decades. To get an integrative view of the sophisticated etiopathogenesis of AD, whole genome transcriptome studies turned out to be indispensable. Here we carried out microarray data collection based on RNA extracted from the retrosplenial cortex and hippocampus of age-matched, eight months old male and female APP/PS1 AD mice and control animals to perform sex- and brain region specific analysis of transcriptome profiles. The results of our studies reveal novel, detailed insight into differentially expressed signature genes and related fold changes in the individual APP/PS1 subgroups. Gene ontology and Venn analysis unmasked that intersectional, upregulated genes were predominantly involved in, e.g., activation of microglial, astrocytic and neutrophilic cells, innate immune response/immune effector response, neuroinflammation, phagosome/proteasome activation, and synaptic transmission. The number of (intersectional) downregulated genes was substantially less in the different subgroups and related GO categories included, e.g., the synaptic vesicle docking/fusion machinery, synaptic transmission, rRNA processing, ubiquitination, proteasome degradation, histone modification and cellular senescence. Importantly, this is the first study to systematically unravel sex- and brain region-specific transcriptome fingerprints/signature genes in APP/PS1 mice. The latter will be of central relevance in future preclinical and clinical AD related studies, biomarker characterization and personalized medicinal approaches.

Nuclear ribonucleoprotein RALY downregulates foot-and-mouth disease virus replication but antagonized by viral 3C protease.

The internal ribosome entry site (IRES) element constitutes a cis-acting RNA regulatory sequence that recruits the ribosomal initiation complex in a cap-independent manner, assisted by various RNA-binding proteins and IRES trans-acting factors. Foot-and-mouth disease virus (FMDV) contains a functional IRES element and takes advantage of this element to subvert host translation machinery. Our study identified a novel mechanism wherein RALY, a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family belonging to RNA-binding proteins, binds to the domain 3 of FMDV IRES via its RNA recognition motif residue. This interaction results in the downregulation of FMDV replication by inhibiting IRES-driven translation. Furthermore, our findings reveal that the inhibitory effect exerted by RALY on FMDV replication is not attributed to the FMDV IRES-mediated assembly of translation initiation complexes but rather to the impediment of 80S ribosome complex formation after binding with 40S ribosomes. Conversely, 3Cpro of FMDV counteracts RALY-mediated inhibition by the ubiquitin-proteasome pathway. Therefore, these results indicate that RALY, as a novel critical IRES-binding protein, inhibits FMDV replication by blocking the formation of 80S ribosome, providing a deeper understanding of how viruses recruit and manipulate host factors. IMPORTANCE: The translation of FMDV genomic RNA driven by IRES element is a crucial step for virus infections. Many host proteins are hijacked to regulate FMDV IRES-dependent translation, but the regulatory mechanism remains unknown. Here, we report for the first time that cellular RALY specifically interacts with the IRES of FMDV and negatively regulates viral replication by blocking 80S ribosome assembly on FMDV IRES. Conversely, RALY-mediated inhibition is antagonized by the viral 3C protease by the ubiquitin-proteasome pathway. These results would facilitate further understanding of virus-host interactions and translational control during viral infection.

A tumor suppressor protein encoded by circKEAP1 inhibits osteosarcoma cell stemness and metastasis by promoting vimentin proteasome degradation and activating anti-tumor immunity.

BACKGROUND: Osteosarcoma (OS) is one of most commonly diagnosed bone cancer. Circular RNAs (circRNAs) are a class of highly stable non-coding RNA, the majority of which have not been characterized functionally. The underlying function and molecular mechanisms of circRNAs in OS have not been fully demonstrated. METHOD: Microarray analysis was performed to identify circRNAs that are differentially-expressed between OS and corresponding normal tissues. The biological function of circKEAP1 was confirmed in vitro and in vivo. Mass spectrometry and western blot assays were used to identify the circKEAP1-encoded protein KEAP1-259aa. The molecular mechanism of circKEAP1 was investigated by RNA sequencing and RNA immunoprecipitation analyses. RESULTS: Here, we identified a tumor suppressor circKEAP1, originating from the back-splicing of exon2 of the KEAP1 gene. Clinically, circKEAP1 is downregulated in OS tumors and associated with better survival in cancer patients. N6-methyladenosine (m6A) at a specific adenosine leads to low expression of circKEAP1. Further analysis revealed that circKEAP1 contained a 777 nt long ORF and encoded a truncated protein KEAP1-259aa that reduces cell proliferation, invasion and tumorsphere formation of OS cells. Mechanistically, KEAP1-259aa bound to vimentin in the cytoplasm to promote vimentin proteasome degradation by interacting with the E3 ligase ARIH1. Moreover, circKEAP1 interacted with RIG-I to activate anti-tumor immunity via the IFN-γ pathway. CONCLUSION: Taken together, our findings characterize a tumor suppressor circKEAP1 as a key tumor suppressor regulating of OS cell stemness, proliferation and migration, providing potential therapeutic targets for treatment of OS.

Proteasome condensate formation is driven by multivalent interactions with shuttle factors and ubiquitin chains.

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin-binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome-Rpn1, Rpn10, and Rpn13-and the Ubl domains of Rad23 and Dsk2 are critical under different condensate-inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes.

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

BACKGROUND: Proteasome inhibition is a promising strategy for cancer therapy. Bortezomib, which primarily targets the chymotrypsin-like activity of PSMB5, has demonstrated efficacy in various tumors. However, there is variable sensitivity to bortezomib, which could be attributed, in part, to variations in the expression of proteasome subunits. METHODS AND RESULTS: In this study, we investigated whether miR-383 affects the expression of proteasome subunits in osteosarcoma (OS) cells, and if so, whether OS cells display differential sensitivity to bortezomib concerning miR-383 expression. We detected a decreased miR-383 expression in OS cells and tissues. Then we found a negative correlation between the cytotoxicity of bortezomib and the expression level of the proteasome 20S core particle subunit ß5 (PSMB5). Intriguingly, we identified PSMB5 as a direct target of miR-383. Increased expression of miR-383 resulted in decreased PSMB5 expression and increased sensitivity to bortezomib in OS cells. CONCLUSIONS: In summary, our findings present the initial comprehensive analysis of the function of miR-383 in OS. The outcomes indicate that miR-383 may augment the anticancer effect of bortezomib through PSMB5 repression, offering a novel therapeutic approach in OS and a fresh pathway for proteasome regulation.

Acute accumulation of PIM2 and NRF2 and recovery of ß5 subunit activity mitigate multiple myeloma cell susceptibility to proteasome inhibitors.

Resistance to proteasome inhibitors (PIs) has emerged as an important clinical issue. We investigated the mechanisms underlying multiple myeloma (MM) cell resistance to PIs. To mimic their pharmacokinetic/pharmacodynamic (PK/PD) profiles, MM cells were treated with bortezomib and carfilzomib for 1 h at concentrations up to 400 and 1,000 nM, respectively. Susceptibility to these PIs markedly varied among MM cell lines. Pulsatile treatments with PIs suppressed translation, as demonstrated by incorporation of puromycin at 24 h in PI-susceptible MM.1S cells, but not PI-resistant KMS-11 cells. Inhibition of ß5 subunit activity decreased at 24 h in KMS-11 cells, even with the irreversible PI carfilzomib, but not under suppression of protein synthesis with cycloheximide. Furthermore, the proteasome-degradable pro-survival factors PIM2 and NRF2 acutely accumulated in MM cells subjected to pulsatile PI treatments. Accumulated NRF2 was trans-localized into the nucleus to induce the expression of its target gene, HMOX1, in MM cells. PIM and Akt inhibition restored the anti-MM effects of PIs, even against PI-resistant KMS-11 cells. Collectively, these results suggest that increased synthesis of ß5 proteasome subunit and acute accumulation of PIM2 and NRF2 reduce the anti-MM effects of PIs.

In vivo evidence for homeostatic regulation of ribosomal protein levels in Drosophila.

The ribosome is a molecular machine essential for protein synthesis, which is composed of approximately 80 different ribosomal proteins (Rps). Studies in yeast and cell culture systems have revealed that the intracellular level of Rps is finely regulated by negative feedback mechanisms or ubiquitin-proteasome system, which prevents over- or under-abundance of Rps in the cell. However, in vivo evidence for the homeostatic regulation of intracellular Rp levels has been poor. Here, using Drosophila genetics, we show that intracellular Rp levels are regulated by proteasomal degradation of excess Rps that are not incorporated into the ribosome. By establishing an EGFP-fused Rp gene system that can monitor endogenously expressed Rp levels, we found that endogenously expressed EGFP-RpS20 or -RpL5 is eliminated from the cell when RpS20 or RpL5 is exogenously expressed. Notably, the level of endogenously expressed Hsp83, a housekeeping gene, was not affected by exogenous expression of Hsp83, suggesting that the strict negative regulation of excess protein is specific for intracellular Rps. Further analyses revealed that the maintenance of cellular Rp levels is not regulated at the transcriptional level but by proteasomal degradation of excess free Rps as a protein quality control mechanism. Our observations provide not only the in vivo evidence for the homeostatic regulation of Rp levels but also a novel genetic strategy to study in vivo regulation of intracellular Rp levels and its role in tissue homeostasis via cell competition.Key words: ribosomal protein, proteasomal degradation, Drosophila.

Studying Antifatigue Mechanism of Tyr-Pro-Leu-Pro in Exercise Mice Using Label-Free Proteomics.

In our previous study, yeast-derived peptide Tyr-Pro-Leu-Pro (YPLP) was found to prolong treadmill time and relieve muscle fatigue in ICR mice. The present study aimed to further investigate the antifatigue mechanism of YPLP. Three doses of YPLP (10, 25, and 50 mg/kg·d) were given to exercise mice for 4 weeks. Results showed that YPLP reduced the oxidative response via the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway and promoted energy metabolism through the AMP-activated protein kinase (AMPK) pathway. Label-free proteomics results showed that 81 differential abundance proteins (DAPs) were regulated by high-dose YPLP. These DAPs belonged to proteasome, mitochondrial, and muscle proteins. YPLP was mainly involved in proteasome, aminoacyl-tRNA biosynthesis, focal adhesion, and MAPK signal pathways to enhance muscle endurance. Furthermore, real-time quantitative PCR and Western blotting results proved that YPLP upregulated Psmd14 expression and downregulated p38 MAPK expression. Overall, this study revealed the mechanism behind YPLP to alleviate exercise fatigue.

Ubiquitin-proteasome system regulation of a key gene regulatory factor, Paf1C.

Paf1 (Polymerase-associated factor 1) complex (Paf1C) is evolutionarily conserved from yeast to humans, and facilitates transcription elongation as well as co-transcriptional histone covalent modifications and mRNA 3'-end processing. Thus, Paf1C is a key player in regulation of eukaryotic gene expression. Paf1C consists of Paf1, Cdc73, Ctr9, Leo1 and Rtf1 in both yeast and humans, but it has an additional component, Ski8, in humans. The abundances of these components regulate the assembly of Paf1C and/or its functions, thus implying the mechanisms involved in regulating the abundances of the Paf1C components in altered gene expression and hence cellular pathologies. Towards finding the mechanisms associated with the abundances of the Paf1C components, we analyzed here whether the Paf1C components are regulated via targeted ubiquitylation and 26S proteasomal degradation. We find that the Paf1C components except Paf1 do not undergo the 26S proteasomal degradation in both yeast and humans. Paf1 is found to be regulated by the ubiquitin-proteasome system (UPS) in yeast and humans. Alteration of such regulation changes Paf1's abundance, leading to aberrant gene expression. Intriguingly, while the Rtf1 component of Paf1C does not undergo the 26S proteasomal degradation, it is found to be ubiquitylated, suggesting that Rtf1 ubiquitylation could be engaged in Paf1C assembly and/or functions. Collectively, our results reveal distinct UPS regulation of the Paf1C components, Paf1 and Rtf1, in a proteolysis-dependent and -independent manners, respectively, with functional implications.

Development of an ubiquitin-proteasome system signature for predicting prognosis and providing therapeutic guidance for patients with triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a pathological subtype with a high mortality, and the development of inhibitors in the ubiquitin-proteasome system (UPS) component could be a novel therapeutic tool. METHODS: Triple-negative breast cancer data were obtained from The Cancer Genome Atlas (TCGA), and subtype analysis was performed by consistent clustering analysis to identify molecular subtypes of TNBC according to UPS characteristics. Differential analysis, COX and least absolute shrinkage and selection operator (LASSO) COX regression analyses were performed to select genes associated with overall survival in TNBC. The final prognostic model (UPS score) was determined using the LASSO COX model. The model performance was assessed using receiver operating characteristic (ROC) curves and survival curves. In addition, the results of the UPS score on analyzing the abundance of immune cell infiltration and immunotherapy were explored. Finally, we developed a nomogram for TNBC survival prediction. RESULTS: Two UPS subtypes (UPSMS1 and UPSMS2) showing significant survival differences were classified. COX regression analysis on differentially expressed genes in UPSMS1 and UPSMS2 filtered five genes that affected overall survival. Based on the regression coefficients and expression data of the five genes, we built a prognostic assessment system (UPS score). The UPS score showed consistent prognostic and therapeutic guidance values. Finally, the ROC curve of the nomogram and UPS score showed the highest predictive efficacy compared with traditional clinical prognostic indicators. CONCLUSION: The UPS score represented a promising prognostic tool to predict overall survival and immune status and guide personalized treatment selection in TNBC patients, and this study may provide a more practical alternative for clinical monitoring and management of TNBC.

Comprehensive analysis of PSMD family members and validation of PSMD9 as a potential therapeutic target in human glioblastoma.

AIMS: PSMD family members, as important components of the 26S proteasome, are well known to be involved in protein degradation. However, their role in glioblastoma (GBM) has not been rigorously investigated. We aimed to perform systematic analysis of the expression signature, prognostic significance and functions of PSMD family genes in GBM to reveal potential prognostic markers and new therapeutic targets among PSMD family members. METHODS: In this study, we systemically analyzed PSMD family members in terms of their expression profiles, prognostic implications, DNA methylation levels, and genetic alterations; the relationships between their expression levels and immune infiltration and drug sensitivity; and their potential functional enrichment in GBM through bioinformatics assessment. Moreover, in vitro and in vivo experiments were used to validate the biological functions of PSMD9 and its targeted therapeutic effect in GBM. RESULTS: The mRNA levels of PSMD5/8/9/10/11/13/14 were higher in GBM than in normal brain tissues, and the mRNA levels of PSMD1/4/5/8/9/11/12 were higher in high-grade glioma (WHO grade III & IV) than in low-grade glioma (WHO grade II). High mRNA expression of PSMD2/6/8/9/12/13/14 and low mRNA expression of PSMD7 were associated with poor overall survival (OS). Multivariate Cox regression analysis identified PSMD2/5/6/8/9/10/11/12 as independent prognostic factors for OS prediction. In addition, the protein-protein interaction network and gene set enrichment analysis results suggested that PSMD family members and their interacting molecules were involved in the regulation of the cell cycle, cell invasion and migration, and other biological processes in GBM. In addition, knockdown of PSMD9 inhibited cell proliferation, invasion and migration and induced G2/M cell cycle arrest in LN229 and A172 GBM cells. Moreover, PSMD9 promoted the malignant progression of GBM in vivo. GBM cell lines with high PSMD9 expression were more resistant to panobinostat, a potent deacetylase inhibitor, than those with low PSMD9 expression. In vitro and in vivo experiments further validated that PSMD9 overexpression rescued the GBM inhibitory effect of panobinostat. CONCLUSION: This study provides new insights into the value of the PSMD family in human GBM diagnosis and prognosis evaluation, and we further identified PSMD9 as a potential therapeutic target. These findings may lead to the development of effective therapeutic strategies for GBM.