Department of Pathology and Diagnostics, Section of Immunology, University of Verona, Policlinico G. B. Rossi, P. le L. Scuro 10, 37134 Verona, Italy.
J Biol Chem. 2012 Mar 2;287(10):7367-73. doi: 10.1074/jbc.M111.316828. Epub 2012 Jan 6.
Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.
2 型核糖体失活蛋白（RIPs）家族的成员（如蓖麻毒素、相思豆毒素）是具有强大细胞毒性的强效细胞毒素，对真核细胞表现出很强的致死活性。2 型 RIP 包含两条通过二硫键连接的多肽链（通常命名为 A，代表“活性”，和 B，代表“结合”）。细胞中毒是一个还原过程的结果，在这个过程中，毒域通过内质网（ER）腔中的氧化还原酶从结合域中切割出来。2 型 RIP 中最著名的例子是蓖麻毒素。已经证明蛋白二硫键异构酶（PDI）参与了蓖麻毒素的还原过程；然而，当 PDI 从细胞级分制备物中耗尽时，蓖麻毒素的还原仍能发生，这表明其他氧化还原酶也可能参与这一过程。我们研究了 TMX 在 2 型 RIPs 蓖麻毒素和相思豆毒素细胞中毒过程中的作用，TMX 是 PD1 家族的一种跨膜硫氧还蛋白相关蛋白。在 A549 细胞中过表达 TMX 会导致细胞毒性显著增加，与对照 Mock 处理的细胞相比，蓖麻毒素或相思豆毒素的细胞毒性增加。相反，在用 saporin 或 Pseudomonas exotoxin A 处理 A549 细胞或对照细胞后，没有观察到细胞毒性的差异，这两种毒素的细胞内激活机制不依赖于还原（saporin）或仅部分依赖于还原（Pseudomonas exotoxin A）。此外，在前列腺细胞系 DU145 中沉默 TMX 进一步降低了细胞对蓖麻毒素中毒的敏感性，这进一步证实了该酶在细胞内蓖麻毒素激活中的作用。
