The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol
The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol
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DOI：
10.1021/bi011580v
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发表时间：
2002-03-12
期刊：
影响因子：
2.9
通讯作者：
Lord, JM
中科院分区：
文献类型：
--
作者：
Deeks, ED;Cook, JP;Lord, JM
包括蓖麻毒素 A 链 (RTA) 在内的多种蛋白质毒素通过内吞作用进入哺乳动物细胞，随后通过内质网 (ER) 膜易位到达其胞质底物。为了实现这种输出，此类毒素利用 ER 相关蛋白降解 (ERAD) 途径，但必须至少部分地逃避 ERAD 底物的正常降解命运。从内质网转移的毒素的赖氨酸含量异常低。由于赖氨酰残基是潜在的泛素化位点，因此有人提出赖氨酸的缺乏会降低泛素化和随后泛素介导的蛋白酶体降解的机会[Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]。在这里，我们为这一假设提供了实验支持。 RTA 内的两个赖氨酰残基更改为精氨酰残基。它们在 RTA 中的替换没有显着的稳定作用，表明内源赖氨酰残基不是泛素附着的常见位点。然而，当以不损害毒素活性、结构或稳定性的方式将四个额外的赖氨酸引入RTA时，降解显着增强。泛素化导致降解增强，使毒素易于被蛋白酶体降解。用蛋白酶体抑制剂 clasto-lactacystin P-内酯处理可将富含赖氨酸的 RTA 的细胞毒性增加到接近野生型蓖麻毒素的水平。与野生型相思豆毒素相比，在第二个核糖体失活蛋白相思豆蛋白 A 链中引入四个额外的赖氨酰残基也显着降低了全毒素的细胞毒性。这种效应也可以通过蛋白酶体抑制来逆转。我们的数据支持这样的假设：低赖氨酸含量的进化是从内质网逆转位的毒素的避免降解策略。
Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin P-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.