Protein folding in the endoplasmic reticulum

内质网中的蛋白质折叠

• 批准号: RGPIN-2014-04686
• 负责人: Gehring, Kalle
• 金额: $ 3.86万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629826
• 关键词: Protein; folding; endoplasmic; reticulum

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 In unpublished work, we have cloned, expressed and purified calnexin cycle components from yeast and a key ER sensor of unfolded proteins.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.

膜和分泌蛋白通过由内质网（ER）、高尔基体和分泌囊泡组成的分泌途径获得翻译后修饰并折叠。为了实现这一目标，细胞进化出一套专门的伴侣、酶和受体分子，介导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募集之间的联系，以及2)未折叠蛋白质的识别机制。这两个过程都是由钙连接素周期的伴侣进行的。calnexin循环由折叠糖蛋白的伴侣蛋白和修饰附着的聚糖以反映蛋白质折叠状态的酶组成。这个循环的功能是促进新合成的糖蛋白的有效折叠，防止它们过早地从内质网输出。关于calnexin周期有许多未解之谜：1)伴侣蛋白在低等生物中是否有类似的功能？ii)钙连蛋白周期如何区分折叠蛋白和未折叠蛋白？iii)未折叠的蛋白质是如何被识别的，是否有一个通用的编码？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，我们发现了肽基脯氨酸异构酶与钙连联蛋白周期之间的一种新的关联。我们还确定了伴侣钙网蛋白如何识别聚糖。在未发表的工作中，我们从酵母中克隆、表达和纯化了calnexin循环成分和未折叠蛋白的关键内质网传感器。在此，我建议将结构生物学和体外功能分析相结合，继续进行这些研究，重点研究两个目标：1)酵母凝集素伴侣复合物的结构和功能研究。我们已经从酵母钙连蛋白（Cne1p）中鉴定出相互作用环，并表明它与酵母蛋白二硫异构酶（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复合物的共结晶。我们将进行功能分析来验证我们的假设，即Cne1p•Mpd1p的功能类似于它们的哺乳动物同源物。这项工作将把我们对calnexin循环的理解扩展到酵母er的良好表征2)udp -葡萄糖：糖蛋白-葡萄糖基转移酶（UGGT）的研究。这种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链聚糖上。我们有从多个物种中纯化ugt的大量初步数据，并进行了功能分析，表明纯化的蛋白是有活性的。自提交意向书以来，我们在电子显微镜（EM）上取得了令人兴奋的进展。ugt的阴性染色三维重建可再现地显示一个大的中心空腔，我们假设其中包含催化位点。这可以解释ugt对未折叠蛋白的特异性。折叠结构域上的聚糖无法进入催化位点，而未折叠多肽链上的聚糖则能够进入催化腔室。疏水残基的存在将有利于未折叠的蛋白质片段的结合，并进一步增加酶的选择性。我们将通过ugt的EM， x射线晶体学和SAXS研究来验证这一假设。我的团队有能力在了解内质网蛋白折叠方面取得实质性进展。我们在提出的技术、获得所需的质粒、材料和测定方面有经验，并与EM和ER伴侣专家建立了合作关系。这项研究促进了生物学、化学和物理学交叉领域的跨学科训练。