Protein folding in the endoplasmic reticulum

膜蛋白和分泌蛋白获得翻译后修饰，并通过内质网(ER)、高尔基体和分泌小泡组成的分泌途径折叠。为了做到这一点，细胞已经进化出一套专门的伴侣、酶和受体分子，这些分子介导了蛋白质折叠和运输的多个步骤。 我的研究涉及内质网中蛋白质折叠的两个方面：1)N-连接糖蛋白的碳水化合物结构与伴侣蛋白招募之间的联系；2)未折叠蛋白质的识别机制。这两个过程都是由钙粘连蛋白循环的伴侣进行的。钙粘连蛋白循环由折叠糖蛋白的伴侣和修饰连接的多糖以反映蛋白质的折叠状态的酶组成。该循环的功能是促进新合成的糖蛋白的有效折叠，并防止它们过早地从内质网输出。 关于Calnexin循环还有许多悬而未决的问题：i)伴侣蛋白在低等生物体中的作用类似吗？Ii)Calnexin循环如何区分折叠和未折叠的蛋白质？Iii)对于如何识别未折叠的蛋白质，有没有一个通用的编码？ 我的研究小组在回答这些问题方面取得了重大进展。在已发表的工作中，我们发现了一个新的联系，在一个肽基脯氨酰异构酶和钙粘蛋白循环。我们还确定了伴侣钙网硬蛋白识别多聚糖的方式。在未发表的工作中，我们从酵母中克隆、表达和纯化了钙粘连蛋白循环组件和一个关键的未折叠蛋白质的内质网传感器。 在这里，我建议继续这些研究，将结构生物学和体外功能分析结合起来，并专注于两个目标： 1)酵母凝集素伴侣复合体的结构和功能研究。我们已经确定了酵母Calnexin(Cne1p)的相互作用环，并表明它与酵母蛋白二硫键异构酶(Mpd1p)相互作用。我们将识别Mpd1p上的结合表面，并使用该信息来指导络合物的共结晶。我们将进行功能分析来验证我们的假设，即Cne1p·Mpd1p的功能类似于他们的哺乳动物同源基因。这项工作将把我们对Calnexin循环的理解扩展到特性良好的酵母ER。 2)UDP-葡萄糖：糖蛋白葡萄糖转移酶(UGGT)的研究。这个关键的内质网酶专门将葡萄糖残基添加到未折叠蛋白质的N-连接的糖链上。我们有大量的初步数据用于从多个物种中纯化UGGT，功能分析表明纯化的蛋白具有活性。自从提交意向书以来，我们在电子显微镜(EM)方面取得了令人兴奋的进展。UGGT的负染3D重建可重复显示一个大的中心空洞，我们假设该空洞包含催化位置。这就解释了UGGT对未折叠蛋白质的特异性。折叠结构域上的多聚糖不能进入催化部位，而未折叠多肽链上的多聚糖能够进入小室。空腔内疏水残基的存在有利于未折叠蛋白片段的结合，进一步提高了酶的选择性。我们将通过对UGGT的EM、X射线结晶学和SAXS研究来验证这一假说。 我的团队处于有利地位，可以在理解内质网中的蛋白质折叠方面取得实质性进展。我们在所建议的技术方面有经验，获得所需的质粒、材料和分析，并与EM和ER伴侣方面的专家建立了合作关系。这项研究促进了生物、化学和物理之间的跨学科培训。

Membrane and secreted proteins acquire post-translational modifications and become folded through the secretory pathway comprised of the endoplasmic reticulum (ER), the Golgi body and secretory vesicles. To accomplish this, cells have evolved a set of specialized chaperones, enzymes, and receptor molecules that mediate the multiple steps of protein folding and trafficking. My research addresses two aspects of protein folding in the ER: 1) the link between the carbohydrate structure of N-linked glycoproteins and the recruitment of chaperones, and 2) the mechanism of recognition of unfolded proteins. Both processes are carried out by chaperones of the calnexin cycle. The calnexin cycle consists of chaperones that fold glycoproteins and enzymes that modify the attached glycan to reflect the protein's folded state. The function of the cycle is to promote the efficient folding of newly synthesized glycoproteins and prevent their premature export from the ER. There are a number of unanswered questions about the calnexin cycle: i) Do the chaperones function analogously in lower organisms? ii) How does the calnexin cycle distinguish between folded and unfolded proteins? iii) Is there a general code for how unfolded proteins are recognized? My research group has made significant progress in answering these questions. In published work, we identified a novel association between a peptidyl prolyl isomerase and the calnexin cycle. We also determined how the chaperone calreticulin recognizes glycans. In unpublished work, we have cloned, expressed and purified calnexin cycle components from yeast and a key ER sensor of unfolded proteins. Here, I propose to continue these studies by combining structural biology and in vitro functional assays with work focused on two aims: 1) Structural and functional studies of a lectin chaperone complex from yeast. We have identified the interaction loop from yeast calnexin (Cne1p) and shown that it interacts with a yeast protein disulfide isomerase (Mpd1p). We will identify the binding surface on Mpd1p and use that information to guide co-crystallization of the complex. We will carry out functional assays to test our hypothesis that Cne1p•Mpd1p function analogously to their mammalian orthologs. This work will extend our understanding of the calnexin cycle to the well-characterize yeast ER. 2) Studies of UDP-glucose:glycoprotein-glucosyltransferase (UGGT). This key ER enzyme specifically adds a glucose residue to the N-linked glycan of unfolded proteins. We have extensive preliminary data for the purification of UGGT from multiple species with functional assays to show the purified protein is active. Since the submission of the Notice of Intent, we have made exciting progress by electron microscopy (EM). Negative-stain 3D reconstructions of UGGT reproducibly show a large central cavity, which we hypothesize harbors the catalytic site. This would explain the specificity of UGGT for unfolded proteins. Glycans on folded domains are unable to access the catalytic site, while glycans on an unfolded polypeptide chain are able to enter the chamber. The presence of hydrophobic residues lining the cavity would favor the binding of unfolded protein segments and further increase the selectivity of the enzyme. We will test this hypothesis through EM, X-ray crystallography and SAXS studies of UGGT. My group is well-positioned to make substantial progress in understanding protein folding in the ER. We have experience with the techniques proposed, access to the plasmids, materials, and assays required, and established collaborations with experts in EM and ER chaperones. The research promotes interdisciplinary training at the interface of biology, chemistry and physics.