A Luciferase Functional Quantitative Assay for Measuring NF-ĸB Promoter Transactivation Mediated by HTLV-1 and HTLV-2 Tax Proteins

Bergamo, Elisa; Diani, Erica; Bertazzoni, Umberto

doi:10.1007/978-1-4939-6872-5_6

Cited by 3 publications

(3 citation statements)

References 19 publications

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“…pSpCas9(BB)-2A-Puro (PX459) V2.0 vector was purchased from Addgene. NF-κB-Luc and phRG-TK plasmids have been previously described (Bergamo et al, 2017).…”

Section: Methodsmentioning

confidence: 99%

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TRAF3 Is Required for NF-κB Pathway Activation Mediated by HTLV Tax Proteins

et al. 2019

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Human T-cell leukemia viruses type 1 (HTLV-1) and type 2 (HTLV-2) share a common genome organization and expression strategy but have distinct pathological properties. HTLV-1 is the etiological agent of Adult T-cell Leukemia (ATL) and of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), whereas HTLV-2 does not cause hematological disorders and is only sporadically associated with cases of subacute myelopathy. Both HTLV genomes encode two regulatory proteins that play a pivotal role in pathogenesis: the transactivating Tax-1 and Tax-2 proteins and the antisense proteins HBZ and APH-2, respectively. We recently reported that Tax-1 and Tax-2 form complexes with the TNF-receptor associated factor 3, TRAF3, a negative regulator of the non-canonical NF-κB pathway. The NF-κB pathway is constitutively activated by the Tax proteins, whereas it is inhibited by HBZ and APH-2. The antagonistic effects of Tax and antisense proteins on NF-κB activation have not yet been fully clarified. Here, we investigated the effect of TRAF3 interaction with HTLV regulatory proteins and in particular its consequence on the subcellular distribution of the effector p65/RelA protein. We demonstrated that Tax-1 and Tax-2 efficiency on NF-κB activation is impaired in TRAF3 deficient cells obtained by CRISPR/Cas9 editing. We also found that APH-2 is more effective than HBZ in preventing Tax-dependent NF-κB activation. We further observed that TRAF3 co-localizes with Tax-2 and APH-2 in cytoplasmic complexes together with NF-κB essential modulator NEMO and TAB2, differently from HBZ and TRAF3. These results contribute to untangle the mechanism of NF-κB inhibition by HBZ and APH-2, highlighting the different role of the HTLV-1 and HTLV-2 regulatory proteins in the NF-κB activation.

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“…pSpCas9(BB)-2A-Puro (PX459) V2.0 vector was purchased from Addgene. NF-κB-Luc and phRG-TK plasmids have been previously described (Bergamo et al, 2017).…”

Section: Methodsmentioning

confidence: 99%

“…Luciferase functional quantitative assay was performed as previously described (Bergamo et al, 2017). Briefly, HEK293T, TRAF3-KO and Jurkat cells were transfected with 500 ng of the NF-κB-Luc reporter plasmid together with the appropriate expression vectors or an empty vector.…”

Section: Methodsmentioning

confidence: 99%

TRAF3 Is Required for NF-κB Pathway Activation Mediated by HTLV Tax Proteins

et al. 2019

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“…NF-κB luciferase assay was performed according to previously published methods [ 18 ]. 293T cells were seeded in 24-well plate and transfected with NF-κB luciferase reporter gene plasmids (1.5 μg) and Renilla plasmids (0.3 μg) by lipofectamine2000 for 8 h. PF was added at concentrations of 0.001, 0.01, 0.1, 1.0, and 2.5μM overnight.…”

Section: Methodsmentioning

confidence: 99%

Penfluridol targets acid sphingomyelinase to inhibit TNF signaling and is therapeutic against inflammatory autoimmune diseases

et al. 2022

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Background Penfluridol, isolated from an FDA-approved small-molecule drug library as an inhibitor of tumor necrosis factor α (TNFα)-stimulated NF-κB activation, is clinically used to treat chronic schizophrenia and related disorders. This study is aimed to investigate the therapeutic effect of penfluridol on TNFα-stimulated inflammatory autoimmune diseases, particularly inflammatory arthritis. Methods Various in vitro studies to confirm the inhibitory effect of penfluridol on TNFα-induced NF-κB activity in bone marrow-derived macrophages or Raw 264.7 macrophage cell line. In vivo studies assessed the therapeutic effects of penfluridol in various disease models, including TNFα transgenic mice, collagen-induced arthritis, DSS-induced colitis, and TNBS-induced colitis. Identification and characterization of the binding of penfluridol to acid sphingomyelinase using bioinformatics and drug affinity responsive target stability assay. Acid sphingomyelinase activity assays to reveal penfluridol-mediated inhibition of acid sphingomyelinase activity. siRNA knockdown experiments to illustrate the dependence of penfluridol’s anti-TNF activity on acid sphingomyelinase. Results Penfluridol effectively inhibited TNFα-induced NF-κB activation in vitro and alleviated the severity of arthritis and colitis in vivo. Mechanistic studies revealed that penfluridol bound to acid sphingomyelinase and inhibited its activation. In addition, knockdown of acid sphingomyelinase largely abolished the inhibitory effects of penfluridol on TNFα-induced inflammatory cytokine production. Furthermore, penfluridol suppressed the differentiation of spleen naive CD4+T cells to TH1 and TH17 and inhibited M1 macrophage polarization. Conclusion This study provides the rationale for the possible innovative use of penfluridol as a newly identified small-molecule drug for TNFα-driven diseases, such as inflammatory arthritis and colitis.

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