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International Immunopharmacology Jun 2024Diabetic retinopathy (DR), a common complication of diabetes, is characterized by inflammation and neovascularization, and is intricately regulated by the...
Diabetic retinopathy (DR), a common complication of diabetes, is characterized by inflammation and neovascularization, and is intricately regulated by the ubiquitin-proteasome system (UPS). Despite advancements, identifying ubiquitin-related genes and drugs specifically targeting DR remains a significant challenge. In this study, bioinformatics analyses and the Connectivity Map (CMAP) database were utilized to explore the therapeutic potential of genes and drugs for DR. Through these methodologies, flavopiridol was identified as a promising therapeutic candidate. To evaluate flavopiridol's therapeutic potential in DR, an in vitro model using Human Umbilical Vein Endothelial Cells (HUVECs) induced by high glucose (HG) conditions was established. Additionally, in vivo models using mice with streptozotocin (STZ)-induced DR and oxygen-induced retinopathy (OIR) were employed. The current study reveals that flavopiridol possesses robust anti-inflammatory and anti-neovascularization properties. To further elucidate the molecular mechanisms of flavopiridol, experimental validation and molecular docking techniques were employed. These efforts identified DDX58 as a predictive target for flavopiridol. Notably, our research demonstrated that flavopiridol modulates the DDX58/NLRP3 signaling pathway, thereby exerting its therapeutic effects in suppressing inflammation and neovascularization in DR. This study unveils groundbreaking therapeutic agents and innovative targets for DR, and establishes a progressive theoretical framework for the application of ubiquitin-related therapies in DR.
PubMed: 38897127
DOI: 10.1016/j.intimp.2024.112504 -
Molecular Biology Reports Jun 2024MG132, a proteasome inhibitor, is widely used to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity by proteasome-mediated...
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.
Topics: Animals; Leupeptins; Chickens; Carcinoma, Hepatocellular; Cell Line, Tumor; Liver Neoplasms; Promoter Regions, Genetic; Serum Amyloid A Protein; NF-kappa B; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Gene Expression Regulation, Neoplastic
PubMed: 38896168
DOI: 10.1007/s11033-024-09726-9 -
BioRxiv : the Preprint Server For... Jun 2024The proteasome plays a crucial role in cellular homeostasis by degrading misfolded, damaged, or unnecessary proteins. Understanding the regulatory mechanisms of...
The proteasome plays a crucial role in cellular homeostasis by degrading misfolded, damaged, or unnecessary proteins. Understanding the regulatory mechanisms of proteasome activity is vital, particularly the interaction with activators containing the hydrophobic-tyrosine-any amino acid (HbYX) motif. Here, we present ProEnd, a comprehensive database designed to identify and catalog HbYX motif-containing proteins across the tree of life. Using a simple bioinformatics pipeline, we analyzed approximately 73 million proteins from 22,000 reference proteomes in the UniProt/SwissProt database. Our findings reveal the widespread presence of HbYX motifs in diverse organisms, highlighting their evolutionary conservation and functional significance. Notably, we observed an interesting prevalence of these motifs in viral proteomes, suggesting strategic interactions with the host proteasome. As validation two novel HbYX proteins found in this database were tested and found to directly interact with the proteasome. ProEnd's extensive dataset and user-friendly interface enable researchers to explore the potential proteasomal regulator landscape, generating new hypotheses to advance proteasome biology. This resource is set to facilitate the discovery of novel therapeutic targets, enhancing our approach to treating diseases such as neurodegenerative disorders and cancer. Link: http://proend.org/.
PubMed: 38895466
DOI: 10.1101/2024.06.08.598080 -
BioRxiv : the Preprint Server For... Jun 2024Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like...
Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like arthritis, and encephalitis. The host ubiquitin proteasome system (UPS) plays critical roles in regulating cellular processes to control the infections with various viruses, including alphaviruses. Previous studies suggest alphaviruses hijack UPS for virus infection, but the molecular mechanisms remain poorly characterized. In addition, whether certain E3 ubiquitin ligases or deubiquitinases act as alphavirus restriction factors remains poorly understood. Here, we employed a cDNA expression screen to identify E3 ubiquitin ligase TRIM32 as a novel intrinsic restriction factor against alphavirus infection, including VEEV-TC83, SINV, and ONNV. Ectopic expression of TRIM32 reduces alphavirus infection, whereas depletion of TRIM32 with CRISPR-Cas9 increases infection. We demonstrate that TRIM32 inhibits alphaviruses through a mechanism that is independent of the TRIM32-STING-IFN axis. Combining reverse genetics and biochemical assays, we found that TRIM32 interferes with genome translation after membrane fusion, prior to replication of the incoming viral genome. Furthermore, our data indicate that the monoubiquitination of TRIM32 is important for its antiviral activity. Notably, we also show two TRIM32 pathogenic mutants R394H and D487N, related to Limb-girdle muscular dystrophy (LGMD), have a loss of antiviral activity against VEEV-TC83. Collectively, these results reveal that TRIM32 acts as a novel intrinsic restriction factor suppressing alphavirus infection and provides insights into the interaction between alphaviruses and the host UPS.
PubMed: 38895352
DOI: 10.1101/2024.06.04.597282 -
BioRxiv : the Preprint Server For... Jun 2024Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is , encoding a substrate recognition factor of the SCF E3...
Comprehensive single cell transcriptomics analysis of murine osteosarcoma uncovers function in metastasis, genomic instability and immune activation and reveals additional target pathways.
Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is , encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced tumor inflammation. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of and . We showed that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. We further compared this model with OS models with functional disruption of : one with knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We found reduction of T cell exhaustion and upregulation of interferon activation, along with evidence of replicative and endoplasmic reticulum-related stress in the disruption models, and showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased activities of metastasis-related gene signatures in the -disrupted OS, which we validated by observation of a strong reduction in lung metastasis in the knockout mice. Finally, we report several potential mechanisms of escape from targeting in OS, including upregulation of targets, DNA copy number amplification and overexpression of alternative E3 ligase genes, and potential alternative lineage activation. These mechanistic insights into OS tumor biology and function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.
PubMed: 38895216
DOI: 10.1101/2024.06.04.597347 -
Cancers Jun 2024Circulating plasma cells (CPCs) are detected in most multiple myeloma (MM) patients, both at diagnosis and on relapse. A small subset, plasma cell leukemia (PCL),...
Circulating plasma cells (CPCs) are detected in most multiple myeloma (MM) patients, both at diagnosis and on relapse. A small subset, plasma cell leukemia (PCL), represents a different biology and has a poor prognosis. In this retrospective analysis, we evaluated patients with primary (pPCL, n = 35) or secondary (sPCL, n = 49), with ≥5% CPCs and a smaller subset with lower CPCs of 1-4% (n = 20). The median age was 61 years; 45% were men and 54% were Black. High-risk cytogenetics were found in 87% and extramedullary disease in 47%. For the entire cohort, 75% received a proteasome inhibitor, 70% chemotherapy, 54% an immunomodulatory drug, 24% a daratumumab-based regimen and 26% an autologous stem cell transplant (ASCT). The treatments marginally improved the overall survival (OS) for pPCL vs. sPCL (13 vs. 3.5 months = 0.002). However, the 5-year survival for the whole cohort was dismal at 11%. High-risk cytogenetics, low platelets, extramedullary disease and high LDH were independently associated with poor outcomes. Further research is urgently needed to expand the treatment options and improve the outcomes in PCL.
PubMed: 38893268
DOI: 10.3390/cancers16112149 -
Cancers Jun 2024High expression of the receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor () and RTK mutations are associated with high-risk/worse prognosis in...
High expression of the receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor () and RTK mutations are associated with high-risk/worse prognosis in multiple myeloma (MM). Combining the pIGF1R/pINSR inhibitor linsitinib with the proteasome inhibitor (PI) bortezomib seemed promising in a clinical trial, but IGF1R expression was not associated with therapy response. Because the oncogenic impact of mutations is so far unknown, we investigated the functional impact of mutations on survival signaling, viability/proliferation and survival response to therapy. We transfected four human myeloma cell lines (HMCLs) with , and (Sleeping Beauty), generated CRISPR-Cas9 knockouts in the HMCLs U-266 (IGF1R) and L-363 (IGF1R) and tested the anti-MM activity of linsitinib alone and in combination with the second-generation PI carfilzomib in seven HMCLs. knockout entailed reduced proliferation. Upon IGF1R overexpression, survival signaling was moderately increased in all HCMLs and slightly affected by in one HMCL, whereby the viability remained unaffected. Expression of IGF1R reduced pIGF1R-Y1135, especially under serum reduction, but did not impact downstream signaling. Linsitinib and carfilzomib showed enhanced anti-myeloma activity in six out of seven HMCL irrespective of the mutation status. In conclusion, mutations can impact IGF1R activation and/or downstream signaling, and a combination of linsitinib with carfilzomib might be a suitable therapeutic approach for MM patients potentially responsive to IGF1R blockade.
PubMed: 38893258
DOI: 10.3390/cancers16112139 -
International Journal of Molecular... May 2024The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the...
The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the proteasome activator (PA)28 alpha beta (PA28αβ). In particular, several immunogenic peptides have been reported to be PA28αβ-dependent. In contrast, we did not observe a major impact of PA28αβ on the generation of different major histocompatibility complex (MHC) classI ligands. PA28αβ-knockout mice infected with the () or virus showed a normal cluster of differentiation (CD) 8 response and viral clearance. However, we observed that the adoptive transfer of wild-type cells into PA28αβ-knockout mice led to graft rejection, but not vice versa. Depletion experiments showed that the observed rejection was mediated by CD8 cytotoxic T cells. These data indicate that PA28αβ might be involved in the development of the CD8 T cell repertoire in the thymus. Taken together, our data suggest that PA28αβ is a crucial factor determining T cell selection and, therefore, impacts graft acceptance.
Topics: Animals; Graft Rejection; Mice; CD8-Positive T-Lymphocytes; Histocompatibility Antigens Class I; Mice, Knockout; Proteasome Endopeptidase Complex; Ligands; Mice, Inbred C57BL; Lymphocytic choriomeningitis virus; Vaccinia virus
PubMed: 38891837
DOI: 10.3390/ijms25115649 -
International Journal of Molecular... May 2024Cellular senescence is closely related to DNA damage, proteasome inactivity, histone loss, epigenetic alterations, and tumorigenesis. The mammalian proteasome activator... (Review)
Review
Cellular senescence is closely related to DNA damage, proteasome inactivity, histone loss, epigenetic alterations, and tumorigenesis. The mammalian proteasome activator PA200 (also referred to as PSME4) or its yeast ortholog Blm10 promotes the acetylation-dependent degradation of the core histones during transcription, DNA repair, and spermatogenesis. According to recent studies, PA200 plays an important role in senescence, probably because of its role in promoting the degradation of the core histones. Loss of PA200 or Blm10 is a major cause of the decrease in proteasome activity during senescence. In this paper, recent research progress on the association of PA200 with cellular senescence is summarized, and the potential of PA200 to serve as a therapeutic target in age-related diseases is discussed.
Topics: Proteasome Endopeptidase Complex; Cellular Senescence; Humans; Animals; Proteolysis; Histones; Saccharomyces cerevisiae Proteins; Nuclear Proteins
PubMed: 38891826
DOI: 10.3390/ijms25115637 -
Plants (Basel, Switzerland) May 2024In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation....
In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different () gene overexpression plants of . One is transformed with (designated ), driven by the cauliflower mosaic virus promoter, while the other expresses (designated ), driven by the endogenous promoter of . We discovered that and had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both and . Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply.
PubMed: 38891294
DOI: 10.3390/plants13111485