The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.
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DOI：
10.1371/journal.pone.0253260
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Matute-Bello G
中科院分区：
文献类型：
--
作者：
Kajikawa O;Herrero R;Chow YH;Hung CF;Matute-Bello G
我们以前曾报道，可溶性Fas配体（sFasL）的26个氨基酸的N-末端茎区，这是独立于其结合位点，是其生物学功能所必需的。在这里，我们调查的机制，连接的结构与其功能的sFasL茎区。使用定点突变，我们克隆了一种突变形式的sFasL，其中所有的带电荷的氨基酸的茎区被改变为中性丙氨酸（mut-sFasL）。我们使用Fas敏感的Jurkat T细胞系以及小鼠和人肺泡上皮细胞来测试sFasL复合物的生物活性，使用半胱天冬酶-3活性和膜联蛋白-V外化作为读数。最后，我们测试了mut-sFasL对脂多糖诱导的小鼠肺损伤的影响。我们发现，突变的所有8个带电荷的氨基酸的茎区成不带电荷的氨基酸丙氨酸（mut-sFasL）导致降低的凋亡活性相比，野生型sFasL（WT-sFasL）。mut-sFasL减弱了Fas敏感性人T细胞系Jurkat和原代人小气道上皮细胞上的WT-sFasL功能。其抑制机制与mut-sFasL与WT蛋白形成复合物有关。在肠内注射大肠杆菌脂多糖后24小时，肠内注射mut-sFasL导致24小时后炎症反应减弱。因此，sFasL的茎区对生物活性具有关键作用，并且茎区结构的变化可导致在体外和体内干扰野生型蛋白功能的突变变体。
We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.
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DOI：
10.1042/cs20090422
发表时间：
2010-01-26
期刊：
Clinical science (London, England : 1979)
影响因子：
--
作者：
Fudala R;Krupa A;Stankowska D;Allen TC;Kurdowska AK
通讯作者：
Kurdowska AK
DOI：
10.1165/rcmb.2006-0395oc
发表时间：
2007-11-01
影响因子：
6.4
作者：
Krupa, Agnieszka;Walencka, Maria J.;Kurdowska, Anna K.
通讯作者：
Kurdowska, Anna K.
影响因子：
8.8
作者：
Del Sorbo, Lorenzo;Costamagna, Andrea;Ranieri, V. Marco
通讯作者：
Ranieri, V. Marco
影响因子：
6.4
作者：
Matute-Bello, G;Liles, WC;Martin, TR
通讯作者：
Martin, TR
缺乏FAS的小鼠在急性肺损伤模型中损害了肺泡嗜中性粒细胞的募集和抗KC自身抗体的表达降低。
影响因子：
5.8
作者：
Gil S;Farnand AW;Altemeier WA;Gill SE;Kurdowska A;Krupa A;Florence JM;Matute-Bello G
通讯作者：
Matute-Bello G