Collaborative Research: Protein Quality Control in the Endoplasmic Reticulum

当细胞产生用于输出的蛋白质（分泌蛋白）时，至关重要的是这些蛋白质应该是什么样子。如果没有，则有一种质量控制机制，称为内质网相关降解（ERAD），可以检测并破坏异常蛋白质。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），这种细胞反应在极端情况下可能导致程序性细胞死亡，这一事实强调了清除异常蛋白质分泌途径的重要性。Drs。McCracken和Brodsky最初发现ERAD涉及异常蛋白（ERAD底物）的选择，底物蛋白的运输穿过内质网膜进入细胞质，然后通过蛋白酶体对底物蛋白进行蛋白水解降解。这一途径已经被几个实验室证明参与了至少20种不同底物蛋白的降解，并且在从酵母到人类的真核生物物种中是保守的。随后的研究表明，可溶性蛋白底物的ERAD输出需要至少两种er -管腔伴侣，BiP （KAR2）和calnexin。其中之一，BiP，也是蛋白质进入内质网所必需的。Brodsky和McCracken最近在BiP中发现了ERAD特有的突变，作为该项目的一部分，他们将对这些突变（加上他们计划通过位点定向诱变识别或创建的其他突变）进行生物化学表征，以确定ERAD特别需要BiP结构和活性的哪些方面。McCracken和Brodsky也证明了整体膜蛋白CFTR的ERAD途径与可溶性底物蛋白的ERAD途径有本质上的不同，并且涉及一组不同的伴侣蛋白。CFTR降解既不需要BiP也不需要calnexin，但胞质Hsp70伴侣Ssa1p是必需的；相反，可溶性底物蛋白的ERAD不需要Ssa1p。作为这个项目的一部分，我们将探讨这种区别的分子基础。具体来说，我们将使用遗传和生化技术来检验两个假设：(1)Ssa1p是CFTR泛素化所必需的；(2)在溶液中，需要Ssa1p来维持CFTR易于聚集的胞质结构域。多种ERAD底物降解所必需和不可缺少的因素的列表表明，ERAD底物降解的要求可能利用或可能不利用共同因素。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McCracken在酵母中分离出了α - 1蛋白酶抑制剂（A1PiZ） Z变体降解受损的突变。此外，由于内质网中错误折叠蛋白的存在激活了ERAD和UPR，已知的UPR靶基因是A1PiZ降解所必需的。作为该项目的一部分，McCracken和Brodsky将对A1PiZ蛋白水解所需的两类基因进行功能表征；本研究的结果有望为ERAD的选择和靶向过程提供更好的机制理解。总之，这些研究代表了遗传和生化方法的结合，旨在理解细胞生物学中最近发现的细胞途径。该项目将采用多种方法，并将受益于两位合作科学家的协同专业知识。Ardythe McCracken和Jeffrey Brodsky，他们最初发现了ERAD通路。该项目还将继续为本科生和研究生的课堂和实验室研究指导做出贡献。

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.