ER-Localized Chaperones in Toxin Pathogenesis

描述（由申请方提供）：滋贺毒素遵循复杂的细胞内途径以杀死易感细胞。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）。从内质网腔，毒素必须进入细胞质，在那里它酶促灭活28 S核糖体RNA，抑制蛋白质合成。这种最近发现的从内质网腔到细胞质的途径代表了负责降解在ER腔中错误折叠的宿主蛋白的细胞质量控制机制（称为ER相关降解，或ERAD）。滋贺毒素显然“假装”为错误折叠的宿主蛋白，从而利用质量控制途径进入细胞质中的核糖体。在酵母中，已知ERAD需要一个称为Sec 61的孔，以及ER腔内的分子伴侣。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，最近发现霍乱毒素通过Sec 61从ER转运。我们设计了一个遗传筛选的分子参与滋贺毒素贩运和分离的cDNA编码一种新的伴侣。这种分子伴侣，我们命名为HEDJ，被发现定位于ER腔，并与Bip相互作用，Bip是一种已知参与蛋白质易位出ER的分子。我们在这里证明，HEDJ相互作用与滋贺毒素在ER腔毒素运输之前。我们建议，STx，霍乱和其他毒素增选HEDJ和其他分子伴侣跨ER膜运输。然而，与以这种方式运输的宿主分子不同，我们建议STx绕过胞质溶胶内的蛋白体降解。使用在我们的实验室开发的体外测定，这里提出的实验将解决的作用HEDJ和其他分子伴侣在毒素易位到细胞质。连续的免疫共沉淀实验将允许详细解剖的ER-本地化的分子伴侣参与毒素运输。将我们的注意力转向毒素，结构-功能分析将确定STx的哪些部分能够使该分子与分子伴侣相互作用并离开ER。此外，我们将确定是否STx避免蛋白体降解，并将开始检查蛋白体避免的机制。

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.