ectopic expression
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2022 ◽  
Vol 11 ◽  
Author(s):  
Wenjin Qiu ◽  
Xiaomin Cai ◽  
Kaya Xu ◽  
Shibin Song ◽  
Zumu Xiao ◽  
...  

Regenerating liver phosphatase 1 (PRL1) is an established oncogene in various cancers, although its biological function and the underlying mechanisms in glioblastoma multiforme (GBM) remain unclear. Here, we showed that PRL1 was significantly upregulated in glioma tissues and cell lines, and positively correlated with the tumor grade. Consistently, ectopic expression of PRL1 in glioma cell lines significantly enhanced their tumorigenicity and invasion both in vitro and in vivo by promoting epithelial-mesenchymal transition (EMT). Conversely, knocking down PRL1 blocked EMT in GBM cells, and inhibited their invasion, migration and tumorigenic growth. Additionally, PRL1 also stabilized Snail2 through its deubiquitination by activating USP36, thus revealing Snail2 as a crucial mediator of the oncogenic effects of PRL1 in GBM pathogenesis. Finally, PRL1 protein levels were positively correlated with that of Snail2 and predicted poor outcome of GBMs. Collectively, our data support that PRL1 promotes GBM progression by activating USP36-mediated Snail2 deubiquitination. This novel PRL1/USP36/Snail2 axis may be a promising therapeutic target for glioblastoma.


2022 ◽  
Author(s):  
Taeok Bae ◽  
Bohyun Jeong ◽  
Majid Ali Shah ◽  
Eunjung Roh ◽  
Kyeong Kyu Kim ◽  
...  

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway, despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC-deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA-deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway.


2022 ◽  
Vol 12 ◽  
Author(s):  
Katharina Radakovics ◽  
Claire Battin ◽  
Judith Leitner ◽  
Sabine Geiselhart ◽  
Wolfgang Paster ◽  
...  

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.


2022 ◽  
Vol 11 ◽  
Author(s):  
Ting Wen ◽  
Qiao Yi Chen

Canonical histone H3.1 and variant H3.3 deposit at different sites of the chromatin via distinct histone chaperones. Histone H3.1 relies on chaperone CAF-1 to mediate replication-dependent nucleosome assembly during S-phase, while H3.3 variant is regulated and incorporated into the chromatin in a replication-independent manner through HIRA and DAXX/ATRX. Current literature suggests that dysregulated expression of histone chaperones may be implicated in tumor progression. Notably, ectopic expression of CAF-1 can promote a switch between canonical H3.1 and H3 variants in the chromatin, impair the chromatic state, lead to chromosome instability, and impact gene transcription, potentially contributing to carcinogenesis. This review focuses on the chaperone proteins of H3.1 and H3.3, including structure, regulation, as well as their oncogenic and tumor suppressive functions in tumorigenesis.


Author(s):  
Jiazhou Liu ◽  
Xiaoyu Wang ◽  
Jiazheng Sun ◽  
Yuru Chen ◽  
Jie Li ◽  
...  

Breast cancer (BC) is the most common tumor in women, and the molecular mechanism underlying its pathogenesis remains unclear. In this study, we aimed to investigate gene modules related to the phenotypes of BC, and identify representative candidate biomarkers for clinical prognosis of BC patients. Using weighted gene co-expression network analysis, we here identified NPY5R as a hub gene in BC. We further found that NPY5R was frequently downregulated in BC tissues compared with adjacent tumor-matched control tissues, due to its aberrant promoter CpG methylation which was confirmed by methylation analysis and treatment with demethylation agent. Higher expression of NPY5R was closely associated with better prognosis for BC patients. Gene set enrichment analysis showed that transcriptome signatures concerning apoptosis and cell cycle were critically enriched in specimens with elevated NPY5R. Ectopic expression of NPY5R significantly curbed breast tumor cell growth, induced cell apoptosis and G2/M arrest. Moreover, NPY5R also promoted the sensitivity of BC cells to doxorubicin. Mechanistically, we found that NPY5R restricted STAT3 signaling pathway activation through interacting with IL6, which may be responsible for the antitumor activity of NPY5R. Collectively, our findings indicate that NPY5R functions as a tumor suppressor but was frequently downregulated in BC.


2022 ◽  
Vol 11 ◽  
Author(s):  
Yongzhe Li ◽  
Xin Gao

ObjectiveAccumulating evidence has highlighted the roles of long noncoding RNAs (lncRNAs) as competing endogenous RNAs (ceRNAs) of microRNAs (miRNAs) through their binding sites in the progression of glioma. Hereby, we aim to explore the role of LINC00883 as a regulator of miR-136 and its target, NIMA-related kinase 1 (NEK1), thus, its involvement in the drug resistance of glioma cells.Methods and ResultsMechanistic investigations by dual-luciferase reporter, RNA pull-down, and RNA-binding protein immunoprecipitation (RIP) assays indicated that LINC00883 bound to miR-136, thereby blocking miR-136-induced downregulation of NEK1. Through gain-of-function experiments in U251 cells that presented a high drug resistance, we found that ectopic expression of LINC00883 resulted in increased MRP (encoding multidrug resistance-associated protein), limited cell apoptosis, and increased proliferation. Expectedly, depleting LINC00883 yielded tumor-suppressive and anti-chemoresistance effects on U251 cells by increasing miR-136 and inhibiting NEK1. Next, drug-resistant glioma cell line SOWZ1, drug-sensitive glioma cell line SOWZ2, and drug-resistant glioma cell line SOWZ2-BCNU (SOWZ2 cultured in BCNU) were applied to validate the roles of LINC00883 in the regulation of multidrug resistance. LINC00883 knockdown suppressed the viability of SWOZ1, SWOZ2, and SWOZ2-BCNU cells.ConclusionIn conclusion, LINC00883 knockdown reduces drug resistance in glioma. Hence, our study provides a future strategy to prevent drug resistance-induced therapeutic failure in glioma.


Author(s):  
Raul S. Freitas ◽  
Tyler F. Crum ◽  
Kislay Parvatiyar

Corona virus disease 2019 (COVID-19) pathogenesis is intimately linked to the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) and disease severity has been associated with compromised induction of type I interferon (IFN-I) cytokines which coordinate the innate immune response to virus infections. Here we identified the SARS-CoV-2 encoded protein, Spike, as an inhibitor of IFN-I that antagonizes viral RNA pattern recognition receptor RIG-I signaling. Ectopic expression of SARS-CoV-2 Spike blocked RIG-I mediated activation of IFNβ and downstream induction of interferon stimulated genes. Consequently, SARS-CoV-2 Spike expressing cells harbored increased RNA viral burden compared to control cells. Co-immunoprecipitation experiments revealed SARS-CoV-2 Spike associated with interferon regulatory factor 3 (IRF3), a key transcription factor that governs IFN-I activation. Co-expression analysis via immunoassays further indicated Spike specifically suppressed IRF3 expression as NF-κB and STAT1 transcription factor levels remained intact. Further biochemical experiments uncovered SARS-CoV-2 Spike potentiated proteasomal degradation of IRF3, implicating a novel mechanism by which SARS-CoV-2 evades the host innate antiviral immune response to facilitate COVID-19 pathogenesis.


eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Ayana Sawai ◽  
Sarah Pfennig ◽  
Milica Bulajić ◽  
Alexander Miller ◽  
Alireza Khodadadi-Jamayran ◽  
...  

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We found that PRC1 is essential for the specification of segmentally-restricted spinal motor neuron (MN) subtypes, while PRC2 activity is dispensable to maintain MN positional identities during terminal differentiation. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox-dependent specification programs by derepressed Hox13 paralogs (Hoxa13, Hoxb13, Hoxc13, Hoxd13). These results indicate that PRC1 can function in the absence of de novo PRC2-dependent histone methylation to maintain chromatin topology and postmitotic neuronal fate.


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