Endoplasmic Reticulum Associated Degradation(ERAD)of Membrane Proteins in Yeast

 描述（由申请人提供）：新合成的蛋白质的显著部分易位到内质网（ER）中。一旦与该区室结合，新生多肽被后处理，获得其天然确认，并被分选以递送至其他细胞器或细胞外环境。然而，致病突变可能会损害蛋白质折叠和成熟，这反过来又会产生易于聚集的物种。为了抵消伴随缺陷多肽积累的灾难性影响，可以选择这些底物，将其递送回细胞质，然后通过Brodsky实验室定义的过程降解，称为ER相关降解或ERAD。实验室研究的长期目标是了解疾病相关的ERAD底物是如何被识别和破坏的，以便可以调节该途径中的特定因子或步骤以预防疾病发作。作为实现这一目标的第一步，已经产生了与特定疾病相关的ERAD底物的酵母表达系统。还构建了模型底物以探索如何选择不同类别的异常蛋白质并将其路由用于降解。为了补充这些方法，开发了一种重现膜包埋底物的泛素化的体外测定法。使用这些工具，拟议的研究将首先检查膜蛋白的聚集倾向与其对ERAD的选择之间是否存在直接联系。接下来，将鉴定和表征在ERAD期间维持底物溶解度的膜相关组分。 分泌的蛋白质，交通以外的ER-甚至一些ERAD逃逸-可以捕获和破坏的液泡/溶酶体。参与“决定”晚期分泌途径中的蛋白质是否应该被分选到其最终目的地或应该被递送到液泡/溶酶体的因素定义不清。为了更好地表征这一途径，开发了一个遗传平台。最近的数据表明，α-抑制蛋白家族的成员在ER后降解期间发挥关键作用。将使用生物化学方法对这些蛋白质的功能以及它们与哪些伙伴一起运作进行分子解剖。一种新的细胞表面荧光检测，然后将用于产生互补的，定量的数据质膜驻留时，α-抑制蛋白的活性被改变。这些努力的结果将测试新的假设，导致更深层次的 对蛋白质构象疾病病因学的机械见解，并表征可能作为新的治疗靶点的因素。

 DESCRIPTION (provided by applicant): A significant fraction of newly synthesized proteins translocate into the endoplasmic reticulum (ER). Once associated with this compartment, nascent polypeptides are post- translationally processed, acquire their native confirmations, and are sorted for delivery to other organelles or to the extracellular milieu. However, disease-causing mutations may compromise protein folding and maturation, which in turn generate aggregation-prone species. To off-set the catastrophic effects that accompany the accumulation of defective polypeptides, these substrates can be selected, delivered back to the cytoplasm, and then degraded via a process defined in the Brodsky laboratory and termed ER associated degradation, or ERAD. The long- term goal of research in the laboratory is to understand how disease-associated ERAD substrates are identified and destroyed so that specific factors or steps in this pathway can be modulated to prevent disease onset. As a first step toward this goal, yeast expression systems for ERAD substrates linked to specific maladies have been generated. Model substrates have also been constructed to explore how different classes of aberrant proteins are selected and routed for degradation. To complement these approaches, an in vitro assay that recapitulates the ubiquitination of membrane-embedded substrates was developed. Using these tools, the proposed studies will first examine whether there is a direct link between the aggregation propensity of a membrane protein and its selection for ERAD. Next, the membrane-associated components that maintain substrate solubility during ERAD will be identified and characterized. Secreted proteins that traffic beyond the ER-and even some ERAD escapees-can be captured and destroyed in the vacuole/lysosome. The factors involved in "deciding" whether a protein in the late secretory pathway should be sorted to its final destinations or should be delivered to the vacuole/lysosome are poorly defined. To better characterize this pathway, a genetic platform was developed. Recent data demonstrate that members of the a-arrestin family play a key role during post-ER degradation. A molecular dissection of how these proteins function and with which partners they operate will be undertaken using biochemical methods. A new cell surface fluorescence assay will then be used to generate complementary, quantitative data on plasma membrane residence when a-arrestin activity is altered. The outcome of these efforts will test new hypotheses, lead to deeper mechanistic insights into the etiology of protein conformational diseases, and characterize factors that may serve as novel therapeutic targets.