The transcription factor NF-ĸB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines. Ubiquitination-triggered proteasomal degradation of DNA-bound NF-ĸB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase 7) opposes the activities of E3 ligases, stabilizes DNA-bound NF-ĸB, and thereby promotes NF-ĸB-mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-ĸB ubiquitination and degradation, prevents Toll-like receptor-induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti-inflammatory agents. To this end, we identified an NF-ĸB-binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-ĸB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ĸB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-ĸB activity.

译文

转录因子NF-ĸB是先天免疫反应的主要调节因子,通过介导促炎性细胞因子的表达在炎症疾病中起着重要作用。泛素触发的蛋白酶体降解的DNA结合的NF-κB强烈限制了其靶基因的表达。相反,USP7(去泛素酶泛素特异性肽酶7)对抗E3连接酶的活性,稳定DNA结合的NF-ĸB,从而促进NF-ĸB介导的转录。在膜支持和覆盖分析上使用基因表达和合成肽阵列,我们在这里发现抑制USP7可增加NF-ĸB泛素化和降解,防止Toll样受体诱导的促炎性细胞因子表达,并代表控制炎症的有效策略。但是,USP7在细胞死亡途径,染色质和DNA损伤反应中的广泛调节作用限制了USP7催化抑制剂作为抗炎药的使用。为此,我们在USP7中发现了一个泛素样结构域2的NF- B结合位点,该位点选择性介导了USP7与NF-ĸB亚基的相互作用,但对于与其他蛋白质的相互作用是必不可少的。此外,我们发现USP7中的氨基酸757LDEL760关键地促进了与NF-κB的p65亚基的相互作用。我们的发现支持以下观点:通过开发这种去泛素酶的非催化抑制剂来消除NF-κB活性,USP7活性可能以底物选择性的方式靶向。

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