Collaborative Research: Protein Quality Control in the Endoplasmic Reticulum

当细胞制造蛋白质用于输出（分泌蛋白质）时，蛋白质是它们应该是的是至关重要的。 如果没有，则有一种称为内质网相关降解（ERAD）的质量控制机制，可以检测异常蛋白质并将其破坏。 如果错误折叠的蛋白质在内质网（ER）中积累，它们会诱导“未折叠蛋白质反应”（UPR），这是一种在极端情况下可能导致程序性细胞死亡的细胞反应，这一事实强调了清除异常蛋白质分泌途径的重要性。 麦克拉肯和Brodsky博士最初发现ERAD涉及异常蛋白（ERAD底物）的选择，底物蛋白穿过ER膜返回细胞质的运输，以及随后通过蛋白酶体对底物蛋白的蛋白水解降解。 此后，几个实验室已经证明该途径参与至少20种不同底物蛋白的降解，并且在从酵母到人类的真核物种中是保守的。 随后的工作表明，至少两个ER-内腔分子伴侣，BiP（KAR 2）和钙连接蛋白，需要ERAD输出可溶性蛋白质底物。 其中之一BiP也是蛋白质输入ER所必需的。 Brodsky和麦克拉肯最近发现了ERAD特异性的BiP突变，作为该项目的一部分，他们将对这些突变进行生物化学表征（以及他们计划通过定点诱变识别或创建的其他突变），以确定ERAD特别需要BiP结构和活性的哪些方面。 麦克拉肯和Brodsky还证明了整合膜蛋白CFTR的ERAD途径与可溶性底物蛋白的ERAD途径有很大不同，并且涉及不同的伴侣蛋白。 CFTR降解既不需要BiP也不需要钙连接蛋白，但胞质Hsp 70伴侣Ssa 1 p是必需的;相反，Ssa 1 p不需要可溶性底物蛋白的ERAD。 作为该项目的一部分，将探索这种区别的分子基础。 具体而言，我们将使用遗传学和生物化学技术来验证两个假设：（1）Ssa 1 p是CFTR泛素化所必需的;（2）Ssa 1 p是维持CFTR在溶液中易于聚集的胞质结构域所必需的。因此，继续鉴定特定底物周转所需的基因是必不可少的。为此，Brodsky和麦克拉肯已经分离出突变，其中α 1-蛋白酶抑制剂（A1 PiZ）的Z变体的降解在酵母中受到损害。此外，由于ER中错误折叠蛋白的存在激活ERAD和UPR，因此已经鉴定了A1 PiZ降解所需的已知UPR靶基因。作为该项目的一部分，麦克拉肯和Brodsky将对A1 PiZ蛋白水解所需的两类基因进行功能表征;这项研究的结果有望提供对ERAD选择和靶向过程更好的机制理解。总之，这些研究代表了遗传和生化方法的组合，旨在理解最近发现的细胞生物学细胞途径。 该项目将采用多种方法，并将受益于最初发现ERAD途径的两位合作科学家Ardythe麦克拉肯和Jeffrey Brodsky博士的协同专业知识。 该项目还将继续为本科生和研究生的课堂和实验室研究教学做出贡献。

When cells make proteins for export (secretory proteins), it is critically important that the proteins are as they should be. If not, there is a quality control mechanism, termed Endoplasmic Reticulum-Associated Degradation (ERAD) that detects aberrant proteins and destroys them. The importance of cleansing the secretory pathway of aberrant proteins is underscored by the fact that if mis-folded proteins accumulate in the endoplasmic reticulum (ER), they induce the "unfolded protein response" (UPR), a cellular response that can lead in extreme cases to programmed cell death. Drs. McCracken and Brodsky originally discovered that ERAD involves the selection of aberrant proteins (ERAD substrates), transport of the substrate proteins back across the ER membrane into the cytoplasm, and subsequent proteolytic degradation of the substrate proteins via the proteasome. This pathway has since been shown by several laboratories to be involved in the degradation of at least 20 different substrate proteins and to be conserved across eukaryotic species from yeast to humans. Subsequent work demonstrated that at least two ER-lumenal chaperones, BiP (KAR2) and calnexin, are required for ERAD export of soluble protein substrates. One of these, BiP, is also required for protein import into the ER. Brodsky and McCracken have recently identified mutations in BiP that are specific for ERAD, and as part of this project they will biochemically characterize these mutations (plus others that they plan to identify or create via site-directed mutagenesis) in order to determine what aspects of BiP structure and activity are specifically required for ERAD. McCracken and Brodsky have also demonstrated that the ERAD pathway for an integral membrane protein, CFTR, is substantially different from that for soluble substrate proteins and involves a different set of chaperones. Neither BiP nor calnexin are required for CFTR degradation, but a cytosolic Hsp70 chaperone, Ssa1p, is; conversely, Ssa1p is not required for ERAD of soluble substrate proteins. As part of this project, the molecular basis for this distinction will be explored. Specifically, two hypotheses will be examined using genetic and biochemical techniques: (1) Ssa1p is required for CFTR ubiquitination; and (2) Ssa1p is required to maintain an aggregation-prone cytoplasmic domain of CFTR in solution.A tabulation of the factors necessary and dispensable for the degradation of multiple ERADsubstrates indicates that the requirements for the degradation of ERAD substrates may or may notutilize common factors. Thus, the continued identification of genes required for the turnover of agiven substrate is essential. To this end, Brodsky and McCracken have isolated mutations in which the degradation of the Z variant of Alpha1-Protease Inhibitor (A1PiZ) is compromised in yeast. In addition, because the presence of mis-folded proteins in the ER activate both ERAD and the UPR, known UPR-target genes that are required for the degradation of A1PiZ have been identified. As part of this project, McCracken and Brodsky will carry out a functional characterization of both classes of genes necessary for the proteolysis of A1PiZ; results from this study are expected to provide a better mechanistic understanding of the ERAD selection and targeting process.In sum, these studies represent a combination of genetic and biochemical methods aimedtoward understanding a recently discovered cellular pathway in cell biology. The project will employ multiple approaches and will benefit from the synergistic expertise of the two collaborating scientists, Drs. Ardythe McCracken and Jeffrey Brodsky, who initially discovered the ERAD pathway. The project will also continue to contribute to both classroom and laboratory research instruction of undergraduate and graduate students.