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Veterinary Research Jun 2024Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune...
Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune response. The cGAS-STING signalling pathway plays an important role in antiviral innate immunity. However, it remains unclear whether PDCoV achieves immune evasion by regulating the cGAS-STING pathway. Here, we demonstrated that the nonstructural protein 2 (nsp2) encoded by PDCoV inhibits cGAS-STING-mediated type I and III interferon (IFN) responses via the regulation of porcine STING (pSTING) stability. Mechanistically, ectopically expressed PDCoV nsp2 was found to interact with the N-terminal region of pSTING. Consequently, pSTING was degraded through K48-linked ubiquitination and the proteasomal pathway, leading to the disruption of cGAS-STING signalling. Furthermore, K150 and K236 of pSTING were identified as crucial residues for nsp2-mediated ubiquitination and degradation. In summary, our findings provide a basis for elucidating the immune evasion mechanism of PDCoV and will contribute to the development of targets for anti-coronavirus drugs.
Topics: Animals; Swine; Viral Nonstructural Proteins; Deltacoronavirus; Swine Diseases; Membrane Proteins; Coronavirus Infections; Interferon Type I; Immunity, Innate; HEK293 Cells; Immune Evasion; Ubiquitination
PubMed: 38886840
DOI: 10.1186/s13567-024-01330-w -
Biology Direct Jun 2024Long noncoding RNAs (lncRNAs) are implicated in the initiation and progression of diffuse large B-cell lymphoma (DLBCL). Small nucleolar RNA host gene 20 (SNHG20) has...
BACKGROUND
Long noncoding RNAs (lncRNAs) are implicated in the initiation and progression of diffuse large B-cell lymphoma (DLBCL). Small nucleolar RNA host gene 20 (SNHG20) has been recognized as a critical lncRNA in multiple human cancers. However, the role of SNHG20 and its underlying mechanism in DLBCL are still unclear.
METHODS
The expression levels of SNHG20, c-MYC, β-catenin, and ubiquitin-specific peptidase 14 (USP14) were measured by reverse transcription-quantitative polymerase chain reaction (RT‒qPCR) and immunoblotting. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) incorporation, and flow cytometry assays were used to assess the proliferation and apoptosis of DLBCL cells. The transcriptional regulation of SNHG20 by c-MYC was confirmed by a luciferase reporter assay and RNA immunoprecipitation. The interaction between USP14 and β-catenin was demonstrated using coimmunoprecipitation. A subcutaneous xenograft model was constructed to determine the role of SNHG20 in vivo.
RESULTS
In the present study, we found that SNHG20 expression was upregulated in DLBCL cell lines and tissues compared to their normal counterparts. SNHG20 knockdown prominently reduced the proliferation and induced the apoptosis of U2932 and OCI-LY3 cells. However, SNHG20 overexpression increased the proliferation and apoptosis resistance of DLBCL cells. Mechanistically, the expression of SNHG20 was positively regulated by c-MYC in DLBCL cells. C-MYC directly bound to the promoter of SNHG20 to activate its transcription. SNHG20 was expressed mainly in the cytosol in DLBCL cells. SNHG20 silencing did not impact USP14 expression but markedly decreased the level of β-catenin, the substrate of USP14, in DLBCL cells. USP14 overexpression increased the β-catenin level, and this increase was attenuated by SNHG20 knockdown. Treatment with the proteasome inhibitor MG132 abolished SNHG20 knockdown-induced β-catenin downregulation. Moreover, SNHG20 silencing reduced the half-life but increased the ubiquitination of β-catenin in DLBCL cells. SNHG20 knockdown weakened the interaction between both endogenous and exogenous USP14 and β-catenin. In turn, SNHG20 overexpression increased the c-MYC level, and this increase was attenuated by β-catenin knockdown. Importantly, β-catenin knockdown attenuated the SNHG20-mediated increase in DLBCL cell proliferation in vitro and tumour growth in vivo.
CONCLUSIONS
Taken together, our results suggested that c-MYC-activated SNHG20 accelerated the proliferation and increased the apoptosis resistance of DLBCL cells via USP14-mediated deubiquitination of β-catenin. The c-MYC/SNHG20 positive feedback loop may be a new target for anti-DLBCL treatment.
Topics: RNA, Long Noncoding; Humans; beta Catenin; Cell Proliferation; Lymphoma, Large B-Cell, Diffuse; Proto-Oncogene Proteins c-myc; Cell Line, Tumor; Animals; Mice; Ubiquitination; Ubiquitin Thiolesterase; Gene Expression Regulation, Neoplastic; Apoptosis; Mice, Nude
PubMed: 38886753
DOI: 10.1186/s13062-024-00488-9 -
Research Square Jun 2024Microglia are the resident immune cells of the central nervous system (CNS). We and others have shown that the inflammatory response of microglia is partially regulated...
Microglia are the resident immune cells of the central nervous system (CNS). We and others have shown that the inflammatory response of microglia is partially regulated by the immunoproteasome, an inducible form of the proteasome responsible for the generation of major histocompatibility complex (MHC) class I epitopes. While the role of the proteasome in the adaptive immune system is well established, emerging evidence suggests the immunoproteasome may have discrete functions in the innate immune response. Here, we show that inhibiting the immunoproteasome reduces the IFNγ-dependent induction of complement activator C1q, suppresses phagocytosis, and alters the cytokine expression profile in a microglial cell line and microglia derived from human inducible pluripotent stem cells. Moreover, we show that the immunoproteasome regulates the degradation of IκBα, a modulator of NF-κB signaling. Finally, we demonstrate that NADH prevents induction of the immunoproteasome, representing a potential pathway to suppress immunoproteasome-dependent immune responses.
PubMed: 38883799
DOI: 10.21203/rs.3.rs-4467983/v1 -
Frontiers in Molecular Biosciences 2024
PubMed: 38882607
DOI: 10.3389/fmolb.2024.1433721 -
ACS Omega Jun 2024Seed aging is a complicated process influenced by environmental conditions, impacting biochemical processes in seeds and causing deterioration that results in reduced...
Seed aging is a complicated process influenced by environmental conditions, impacting biochemical processes in seeds and causing deterioration that results in reduced viability and vigor. In this study, we investigated the seed aging process of ridge gourd, which is one of the most exported commercial seeds in Thailand using sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 855 proteins were identified among the two groups (0 d/15 d and 0 d/30 d). The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of differentially expressed proteins revealed that in ridge gourd seeds, the aging process altered the abundance of proteins related to the oxidative stress response, nutrient reservoir, and metabolism pathway. The most identified DEPs were mitochondrial proteins, ubiquitin-proteasome system proteins, ribosomal proteins, carbohydrate metabolism-related proteins, and stress response-related proteins. This study also presented the involvement of aconitase and glutathione pathway-associated enzymes in seed aging, with aconitase and total glutathione being determined as possible suggestive biomarkers for aged ridge gourd seeds. This acquired knowledge has the potential to considerably improve growing methods and seed preservation techniques, enhancing seed storage and maintenance.
PubMed: 38882140
DOI: 10.1021/acsomega.4c01270 -
The Journal of Veterinary Medical... Jun 2024The African pygmy hedgehog (Atelerix albiventris) is known to have a high incidence of tumor. However, investigating the tumors of this species has been constrained by...
The African pygmy hedgehog (Atelerix albiventris) is known to have a high incidence of tumor. However, investigating the tumors of this species has been constrained by the limited availability of research materials such as cell lines and genome information. In this study, we successfully established a novel cell line from a histiocytic sarcoma (HS) of an African pygmy hedgehog, allowing us to conduct a drug screening. We investigated using FDA-approved drug library screening to determine which anticancer drug this tumor cell line is sensitive to, and as a result of apoptosis experiments, bortezomib among the three proteasome inhibitors was found to induce cell death of cancer cells by significantly increasing caspase-3 cleavage (P<0.01). Thus, we elucidated that the proteasome inhibitors, particularly bortezomib, exhibit anti-tumor effects on a cell line derived from an HS in an African pygmy hedgehog through a mechanism comparable to that described in human tumors. This study reports the first characterized cell line from the African pygmy hedgehog and also highlights the potential utility of bortezomib as an anti-tumor treatment for HS in this species.
PubMed: 38880614
DOI: 10.1292/jvms.23-0426 -
Brain Research Bulletin Jun 2024The ubiquitin-proteasome system (UPS) controls the majority of protein degradation in cells and dysregulation of the UPS has been implicated in the pathophysiology of...
The ubiquitin-proteasome system (UPS) controls the majority of protein degradation in cells and dysregulation of the UPS has been implicated in the pathophysiology of numerous neurodegenerative disorders, including Alzheimer's disease. Further, strong evidence supports a critical role for the UPS in synaptic plasticity and memory formation. However, while proteasome function is known to decrease broadly in the brain across the lifespan, whether it changes in the hippocampus, a region critical for memory storage and among the first impacted in Alzheimer's disease, at rest and following learning in the aged brain remains unknown. Further, which proteins have altered targeting for protein degradation in the aged hippocampus has yet to be explored and whether learning in advanced age interacts with changes in ubiquitin-proteasome function across the lifespan remains unknown. Here, using proteasome activity assays and unbiased proteomic analyses, we report age-dependent changes in proteasome activity and degradation-specific K48 polyubiquitin protein targeting in the hippocampus and retrosplenial cortex of male and female rats across the lifespan. In the hippocampus, the targets of altered protein degradation were involved in transcription and astrocyte structure or G-protein and Interferon signaling in males and females, respectively. Importantly, we found that contextual fear conditioning led to an increase in proteasome activity and K48 polyubiquitin protein targeting in the hippocampus of aged male rats, a result in direct contrast to what was previously reported in young adult animals. Together, these data suggest that changes in protein degradation in the hippocampus across the lifespan may be contributing to age-related memory loss.
PubMed: 38879089
DOI: 10.1016/j.brainresbull.2024.111015 -
The Journal of Biological Chemistry Jun 2024Hearing, the ability to sense sounds, and the processing of auditory information are important for perception of the world. Mice lacking expression of neuroplastin (Np),...
Hearing, the ability to sense sounds, and the processing of auditory information are important for perception of the world. Mice lacking expression of neuroplastin (Np), a type-1 transmembrane glycoprotein, display deafness, multiple cognitive deficiencies, and reduced expression of plasma membrane calcium (Ca) ATPases (PMCAs) in cochlear hair cells and brain neurons. In this study, we transferred the deafness causing missense mutations pitch (C315S) and audio-1 (I122N) into human Np (hNp) constructs and investigated their effects at the molecular and cellular level. Computational molecular dynamics show that loss of the disulfide bridge in hNp causes structural destabilization of immunoglobulin-like domain (Ig) III and that the novel asparagine in hNp results in steric constraints and an additional N-glycosylation site in IgII. Additional N-glycosylation of hNp was confirmed by PNGaseF treatment. In comparison to hNp, transfection of hNp and hNp into HEK293T cells resulted in normal mRNA levels but reduced the Np protein levels and their cell surface expression due to proteasomal/lysosomal degradation. Furthermore, hNp and hNp failed to promote exogenous PMCA levels in HEK293T cells. In hippocampal neurons, expression of additional hNp or hNp was less efficient than hNp to elevate endogenous PMCA levels and to accelerate the restoration of basal Ca levels after electrically-evoked Ca transients. We propose that mutations leading to pathological Np variants, as exemplified here by the deafness causing Np mutants, can affect Np-dependent Ca regulatory mechanisms and may potentially cause intellectual and cognitive deficits in humans.
PubMed: 38879011
DOI: 10.1016/j.jbc.2024.107474 -
Cell Stress & Chaperones Jun 2024The evolutionary conserved molecular chaperone heat shock protein 90 (HSP90) plays an indispensable role in tumorigenesis by stabilizing client oncoproteins. Although... (Review)
Review
The evolutionary conserved molecular chaperone heat shock protein 90 (HSP90) plays an indispensable role in tumorigenesis by stabilizing client oncoproteins. Although the functionality of HSP90 is tightly regulated, cancer cells exhibit a unique dependence on this chaperone, leading to its overexpression, which has been associated with poor prognosis in certain malignancies. While various strategies targeting heat shock proteins (HSPs) involved in carcinogenesis have been explored, only inhibition of HSP90 has consistently and effectively resulted in proteasomal degradation of its client proteins. To date, a total of 22 HSP90 inhibitors (HSP90i) have been tested in 186 cancer clinical trials, as reported by clinicaltrials.gov. Among these trials, 60 % have been completed, 10 % are currently active, and 30 % have been suspended, terminated, or withdrawn. HSP90 inhibitors (HSP90i) have been used as single agents or in combination with other drugs for the treatment of various cancer types in clinical trials. Notably, improved clinical outcomes have been observed when HSP90i are used in combination therapies, as they exhibit a synergistic antitumor effect. However, as single agents, HSP90i have shown limited clinical activity due to drug-related toxicity or therapy resistance. Recently, active trials conducted in Japan evaluating TAS-116 (pimitespib) have demonstrated promising results with low toxicity as monotherapy and in combination with the immune checkpoint inhibitor nivolumab. Exploratory biomarker analyses performed in various trials have demonstrated target engagement that suggests the potential for identifying patient populations that may respond favorably to the therapy. In this review, we discuss the advances made in the past 5 years regarding HSP90i and their implications in anticancer therapeutics. Our focus lies in evaluating drug efficacy, prognosis forecast, pharmacodynamic biomarkers, and clinical outcomes reported in published trials. Through this comprehensive review, we aim to shed light on the progress and potential of HSP90i as promising therapeutic agents in cancer treatment.
PubMed: 38878853
DOI: 10.1016/j.cstres.2024.05.005 -
Plant Communications Jun 2024E3 ligases are key enzymes required for protein degradation. Here we identified a C3H2C3 RING domain-containing E3 ubiquitin ligase gene named GhATL68b. It is...
E3 ligases are key enzymes required for protein degradation. Here we identified a C3H2C3 RING domain-containing E3 ubiquitin ligase gene named GhATL68b. It is preferentially and highly expressed in developing cotton fiber cells, and is more conserved in plants than in animals or in archaea. All four orthologs copies of this gene in various diploid cottons and eight in the allotetraploid G. hirsutum were found to originate from a single common ancestor that can be traced back to C. reinhardii at about 992 million years ago (MYA). Structural variations (SVs) occurred in the promoter regions of G. hirsutum, G. herbaceum, G. arboreum and G. raimondii correlated with significantly different methylation patterns. Homozygous CRISPR-Cas9 knock-out cotton lines produced significant poor fiber quality in terms of upper half mean length, elongation at break, uniformity and mature fiber weight. GhATL68b was shown to modulate the homeostasis of 2,4-dienoyl-CoA reductase (GhDECR), a rate-limiting enzyme for β-oxidation of polyunsaturated fatty acids (PUFAs) via the ubiquitin proteasome pathway through in vitro ubiquitination and cell-free protein degradation assays. Fiber cells harvested from these knockout mutants contained significantly lower levels of PUFAs important for glycerophospholipids production and also for plasma membrane fluidity regulations. Finally, the mutant fiber-growth defective phenotype can be fully compensated by adding linolenic acid (C18:3), the most abundant type of PUFA externally in ovule culture media. To our knowledge, this is the first experimentally characterized C3H2C3 type E3 ubiquitin ligase that is involved in regulating fiber cell elongation, and it may thus provide us with a new genetic target for improved cotton lint production.
PubMed: 38877704
DOI: 10.1016/j.xplc.2024.101003