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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• 作者：Emma D. Deeks ; Jonathan P. Cook ; Philip J. Day ; Daniel C. Smith ; Lynne M. Roberts ; and J. Michael Lord
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：March 12, 2002
• 年：2002
• 卷：41
• 期：10
• 页码：3405 - 3413
• 全文大小：139K
• 年卷期：v.41,no.10(March 12, 2002)
• ISSN：1520-4995

文摘

Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells byendocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmicreticulum (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. Toxinsthat translocate from the ER have an unusually low lysine content. Since lysyl residues are potentialubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitinationand subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residueswithin RTA were changed to arginyl residues. Their replacement in RTA did not have a significantstabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitinattachment. However, when four additional lysines were introduced into RTA in a way that did notcompromise 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 es/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-lactone increased the cytotoxicity of thelysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysylresidues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased thecytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomalinhibition. 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.