Role of ER-Localized Chaperones in Toxin Pathogenesis

描述(申请人提供)：志贺毒素遵循一个复杂的细胞内途径，以杀死敏感细胞。在与细胞表面糖脂结合后，毒素被内化并以逆行方式运输到高尔基体和内质网(ER)。从ER腔，毒素必须进入细胞质，在那里它酶促灭活28S核糖体RNA，抑制蛋白质合成。最近发现的从内质网腔到细胞质的这一途径代表了一种细胞质量控制机制，负责降解在内质网管腔中错误折叠的宿主蛋白(称为ER相关降解，简称ERAD)。志贺毒素显然“假装”是一种错误折叠的宿主蛋白，从而利用质量控制途径获得细胞质中的核糖体。在酵母中，ERAD已知需要一个称为Sec61的毛孔，以及内质网管腔内的伴侣。在高等真核生物中，参与这一途径的宿主分子在很大程度上是未知的。然而，最近发现霍乱毒素从急诊室通过Sec61转运。我们设计了一种参与志贺毒素运输的分子的遗传筛选，并分离出编码一种新的伴侣蛋白的cDNAs。这种被我们命名为HEDJ的伴侣被发现定位于内质网管腔，并与Bip相互作用，Bip是一种已知参与蛋白质转运出内质网的分子。我们在这里证明了HEDJ在毒素转运之前与ER管腔中的志贺毒素相互作用。我们认为STX、霍乱和其他毒素选择HEDJ和其他伴侣蛋白通过ER膜运输。然而，与以这种方式运输的宿主分子不同，我们认为STX绕过了胞浆内的蛋白体降解。使用我们实验室开发的体外分析方法，这里提出的实验将研究HEDJ和其他伴侣在毒素转运到细胞质中的作用。连续的免疫共沉淀实验将允许详细剖析参与毒素运输的内质网定位的伴侣。将我们的注意力转向毒素，结构-功能分析将确定STX的哪个部分(S)使分子能够与伴侣相互作用并退出内质网。此外，我们将确定STX是否可以避免蛋白酶体的降解，并将开始研究蛋白酶体避免的机制。

DESCRIPTION (provided by applicant): Shiga toxin follows a complex intracellular pathway in order to kill susceptible cells. After binding to cell surface glycolipids, the toxin is internalized and trafficked in retrograde fashion to the Golgi and endoplasmic reticulum (ER). From the ER lumen, the toxin must gain access to the cytoplasm, where it enzymatically inactivates the 28S ribosomal RNA, inhibiting protein synthesis. This recently discovered pathway from the endoplasmic reticulum lumen to the cytoplasm represents a cellular quality control mechanism responsible for degrading host proteins that misfold in the ER lumen (termed ER-associated degradation, or ERAD). Shiga toxin apparently "pretends" to be a misfolded host protein, and thereby utilizes the quality control pathway to gain access to ribosomes in the cytoplasm. In yeast, ERAD is known to require a pore called Sec61, as well as chaperones within the ER lumen. The host molecules involved in this pathway in higher eucaryotes are largely unknown. Recently, however, cholera toxin was found to be transported from the ER through Sec61. We devised a genetic screen for molecules involved in shiga toxin trafficking and isolated a cDNA encoding a novel chaperone. This chaperone, which we named HEDJ, was found to be localized to the ER lumen and to interact with Bip, a molecule known to be involved in protein translocation out of the ER. We demonstrate here that HEDJ interacts with shiga toxin in the ER lumen prior to toxin transport. We propose that STx, cholera and other toxins co-opt HEDJ and other chaperones for transport across the ER membrane. However, unlike host molecules transported in this manner, we propose that STx bypasses proteosomal degradation within the cytosol. Using in vitro assays developed in our laboratory, the experiments proposed here will address the role of HEDJ and other chaperones in toxin translocation to the cytoplasm. Sequential co-immunoprecipitation experiments will allow a detailed dissection of the ER-localized chaperones involved in toxin transport. Turning our attention to the toxin, structure-function analyses will determine which portion(s) of STx enables the molecule to interact with chaperones and exit the ER. Additionally, we will determine whether STx avoids proteosomal degradation and will begin to examine the mechanism of proteosomal avoidance.