The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity
The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity
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枯草芽孢杆菌 TatAdCd 系统表现出极高水平的底物选择性
DOI：
10.1016/j.bbamcr.2016.10.018
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Frain K
中科院分区：
文献类型：
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作者：
Frain K
尽管人们对 Tat 系统的校对机制知之甚少，但 Tat 系统优先将正确折叠的蛋白质转运穿过细菌膜。大多数研究都集中在革兰氏阴性菌（尤其是大肠杆菌）的 TatABC 系统上，而对革兰氏阳性菌的 TatAC 型系统知之甚少。我们之前已经证明枯草芽孢杆菌 TatAdCd 系统在大肠杆菌中具有功能。大肠杆菌 tatnull 背景，能够运输 TorA-GFP 和天然 TorA（TMAO 还原酶）；在这里，我们检查了它转运带有 TorA 信号序列的其他蛋白质的能力。我们表明，而E。 coliTatABC 运输多种生物治疗药物，包括人生长激素、干扰素 α2b、VH 结构域蛋白和 2 种不同的 scFv，TatAdCd 运输 scFv，但完全排斥其他蛋白质。系统还拒绝两个nativeE。大肠杆菌底物、NrfC 和 FhuD。此外，我们已经证明，TatABC 可以转运多种折叠的 scFv 变体，其表面经过改变以纳入多个盐桥、带电残基（5 个谷氨酸、赖氨酸或精氨酸）或疏水残基（最多 6 个亮氨酸）。相比之下，TatAdCd 完全拒绝许多这些变体，包括那些添加了 5 或 6 个亮氨酸残基的变体。综合数据表明，TatABC 和 TatAdCd 系统具有非常不同的底物选择性，与 E 相比，TatAdCd 系统显示出极高水平的选择性。大肠杆菌系统。该数据还提供了初步建议，即 TatAdCd 可能无法耐受包含疏水性水平高于特定阈值的表面域的基材。
The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especiallyEscherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that theBacillus subtilisTatAdCd system is functional in anE. coli tatnull background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereasE. coliTatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two nativeE. colisubstrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to theE. colisystem. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.