1 Laboratório Nacional de Biociências , LNBio, CNPEM, Campinas, Brazil .
2 Instituto do Coração , Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil .
Antioxid Redox Signal. 2018 Sep 10;29(8):717-734. doi: 10.1089/ars.2017.7297. Epub 2018 Feb 27.
A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17.
Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1 and catalytic site mutant Trx-1 rescued ADAM17 activity, although the interaction with Trx-1 was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1 mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1 mutant showed similar oxidant levels to Trx-1, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity.
We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases.
This unexpected Trx-1 behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.
解整合素金属蛋白酶 17（ADAM17）通过释放 TNFa 和 EGFR 配体等表面蛋白胞外结构域来调节信号事件。我们之前已经将细胞质硫氧还蛋白 1（Trx-1）鉴定为 ADAM17 细胞质结构域的伴侣。然而，Trx-1 调节 ADAM17 的机制尚不清楚，评估 Trx-1 对金属蛋白酶 ADAM17 的影响程度变得至关重要。
通过发现和靶向蛋白质组学方法相结合，我们揭示了 Trx-1 通过直接和间接作用负调控 ADAM17。我们使用合成肽和定点突变进行了基于细胞的测定，并证明了 Trx-1 和 ADAM17 的相互作用界面对于 ADAM17 活性的负调控很重要。然而，Trx-1 和催化位点突变 Trx-1 均挽救了 ADAM17 的活性，尽管与 Trx-1 的相互作用不受影响，这表明 Trx-1 具有间接作用。我们证实，Trx-1 突变体显示出还原能力降低，这通过氧化剂水平解释了对 ADAM17 活性的这种间接影响。有趣的是，尽管 Trx-1 的催化位点得以保留，但 Trx-1 突变体显示出与 Trx-1 相似的氧化剂水平。我们进一步表明，一般的活性氧物质抑制剂 N-乙酰半胱氨酸（NAC）维持了 ADAM17 的调节，依赖于 Trx-1 还原酶活性水平；而电子传递链调节剂鱼藤酮则消除了 Trx-1 对 ADAM17 活性的影响。
我们首次表明，ADAM17 的调节机制，Trx-1 依赖性，可以是通过直接相互作用和间接作用，为异构酶和哺乳动物金属蛋白酶之间的串扰带来了新的见解。
这种出乎意料的 Trx-1 行为是由于更多二聚体的形成，以及随后通过凝胶过滤和质谱分析对二聚体验证、评估的 Trx-1 还原酶活性的降低。抗氧化剂。氧化还原信号。29,717-734。
