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A novel positive feedback-loop between the HTLV-1 oncoprotein Tax and NF-κB activity in T-cells
Retrovirology ( IF 2.7 ) Pub Date : 2020-09-10 , DOI: 10.1186/s12977-020-00538-w
Sebastian Millen 1 , Lina Meretuk 1 , Tim Göttlicher 1 , Sarah Schmitt 1 , Bernhard Fleckenstein 1 , Andrea K Thoma-Kress 1
Affiliation  

Background Human T-cell leukemia virus type 1 (HTLV-1) infects primarily CD4 + T-lymphocytes and evoques severe diseases, predominantly Adult T-Cell Leukemia/ Lymphoma (ATL/L) and HTLV-1-associated Myelopathy/ Tropical Spastic Paraparesis (HAM/TSP). The viral transactivator of the pX region (Tax) is important for initiating malignant transformation, and deregulation of the major signaling pathway nuclear factor of kappa B (NF-κB) by Tax represents a hallmark of HTLV-1 driven cancer. Results Here we found that Tax mutants which are defective in NF-κB signaling showed diminished protein expression levels compared to Tax wildtype in T-cells, whereas Tax transcript levels were comparable. Strikingly, constant activation of NF-κB signaling by the constitutive active mutant of inhibitor of kappa B kinase (IKK2, IKK-β), IKK2-EE, rescued protein expression of the NF-κB defective Tax mutants M22 and K1-10R and even increased protein levels of Tax wildtype in various T-cell lines while Tax transcript levels were only slightly affected. Using several Tax expression constructs, an increase of Tax protein occurred independent of Tax transcripts and independent of the promoter used. Further, Tax and M22 protein expression were strongly enhanced by 12-O-Tetradecanoylphorbol-13-Acetate [TPA; Phorbol 12-myristate 13-acetate (PMA)]/ ionomycin, inducers of NF-κB and cytokine signaling, but not by tumor necrosis factor alpha (TNF-α). On the other hand, co-expression of Tax with a dominant negative inhibitor of κB, IκBα-DN, or specific inhibition of IKK2 by the compound ACHP, led to a vast decrease in Tax protein levels to some extent independent of Tax transcripts in transiently transfected and Tax-transformed T-cells. Cycloheximide chase experiments revealed that co-expression of IKK2-EE prolongs the half-life of M22, and constant repression of NF-κB signaling by IκBα-DN strongly reduces protein stability of Tax wildtype suggesting that NF-κB activity is required for Tax protein stability. Finally, protein expression of Tax and M22 could be recovered by NH 4 Cl and PYR-41, inhibitors of the lysosome and the ubiquitin-activating enzyme E1, respectively. Conclusions Together, these findings suggest that Tax’s capability to induce NF-κB is critical for protein expression and stabilization of Tax itself. Overall, identification of this novel positive feedback loop between Tax and NF-κB in T-cells improves our understanding of Tax-driven transformation.

中文翻译:

T细胞中HTLV-1癌蛋白税和NF-κB活性之间的新型正反馈回路

背景 人类 T 细胞白血病病毒 1 型 (HTLV-1) 主要感染 CD4 + T 淋巴细胞并引发严重疾病,主要是成人 T 细胞白血病/淋巴瘤 (ATL/L) 和 HTLV-1 相关脊髓病/热带痉挛性下肢轻瘫(火腿/TSP)。pX 区域的病毒反式激活因子 (Tax) 对启动恶性转化很重要,而 Tax 对主要信号通路 kappa B (NF-κB) 核因子的失调代表了 HTLV-1 驱动癌症的标志。结果在这里,我们发现在 NF-κB 信号传导中存在缺陷的 Tax 突变体与 T 细胞中的 Tax 野生型相比,其蛋白质表达水平降低,而 Tax 转录水平相当。引人注目的是,κB 激酶抑制剂(IKK2、IKK-β)、IKK2-EE 的组成型活性突变体持续激活 NF-κB 信号传导,拯救了 NF-κB 缺陷型 Tax 突变体 M22 和 K1-10R 的蛋白质表达,甚至增加了各种 T 细胞系中 Tax 野生型的蛋白质水平,而 Tax 转录物水平仅受到轻微影响。使用几种Tax 表达构建体,Tax 蛋白的增加独立于Tax 转录本和所使用的启动子。此外,Tax 和 M22 蛋白表达被 12-O-Tetradecanoylphorbol-13-Acetate [TPA; Phorbol 12-myristate 13-acetate (PMA)]/ionomycin,NF-κB 和细胞因子信号传导的诱导剂,但不是肿瘤坏死因子 α (TNF-α) 的诱导剂。另一方面,Tax 与 κB 的显性负抑制剂 IκBα-DN 或化合物 ACHP 对 IKK2 的特异性抑制的共表达,导致 Tax 蛋白水平的大幅下降在某种程度上独立于瞬时转染和 Tax 转化的 T 细胞中的 Tax 转录本。放线菌酮追踪实验表明,IKK2-EE 的共表达延长了 M22 的半衰期,并且 IκBα-DN 对 NF-κB 信号的持续抑制强烈降低了 Tax 野生型的蛋白质稳定性,表明 Tax 蛋白需要 NF-κB 活性稳定。最后,分别通过溶酶体抑制剂和泛素激活酶 E1 抑制剂 NH 4 Cl 和 PYR-41 可以恢复 Tax 和 M22 的蛋白质表达。结论 总之,这些发现表明Tax 诱导NF-κB 的能力对于Tax 本身的蛋白质表达和稳定至关重要。全面的,
更新日期:2020-09-10
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