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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611183
• 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 In unpublished work, we have cloned, expressed and purified calnexin cycle components from yeast and a key ER sensor of unfolded proteins.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.

膜和分泌蛋白获得翻译后修饰，并通过由内质网 (ER)、高尔基体和分泌囊泡组成的分泌途径折叠。为了实现这一目标，细胞进化出了一组专门的伴侣、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 ER 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分子伴侣招募之间的联系，2）未折叠蛋白质的识别机制。这两个过程都是由钙联蛋白循环的伴侣进行的。钙连接蛋白循环由折叠糖蛋白的分子伴侣和修饰附着的聚糖以反映蛋白质折叠状态的酶组成。该循环的功能是促进新合成的糖蛋白的有效折叠并防止其过早从内质网输出。 关于钙连接蛋白循环还有许多未解答的问题：i) 分子伴侣在低等生物中是否具有类似的功能？ ii) 钙连接蛋白循环如何区分折叠和未折叠的蛋白质？ iii) 是否有关于如何识别未折叠蛋白质的通用代码？ 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酰异构酶与钙联蛋白循环之间的新关联。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、表达和纯化了钙联蛋白循环成分和未折叠蛋白的关键内质网传感器。 在这里，我建议通过结合结构生物学和体外功能测定来继续这些研究，重点关注两个目标： 1) 酵母凝集素伴侣复合物的结构和功能研究。我们已经鉴定了酵母钙联蛋白 (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复合物的共结晶。我们将进行功能测定来检验我们的假设，即 Cne1p•Mpd1p 的功能与其哺乳动物直系同源物类似。这项工作将把我们对钙联蛋白循环的理解扩展到充分表征的酵母内质网。 2）UDP-葡萄糖：糖蛋白-葡萄糖基转移酶（UGGT）的研究。这种关键的 ER 酶专门将葡萄糖残基添加到未折叠蛋白质的 N 连接聚糖上。我们拥有从多个物种中纯化 UGGT 的大量初步数据，并通过功能测定显示纯化的蛋白质具有活性。自提交意向通知以来，我们在电子显微镜 (EM) 方面取得了令人兴奋的进展。 UGGT 的负染色 3D 重建可重复地显示出一个大的中央空腔，我们假设该空腔包含催化位点。这可以解释 UGGT 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，而未折叠的多肽链上的聚糖则能够进入腔室。腔内衬疏水残基的存在将有利于未折叠蛋白质片段的结合并进一步增加酶的选择性。我们将通过 UGGT 的 EM、X 射线晶体学和 SAXS 研究来检验这一假设。 我的团队处于有利地位，可以在理解内质网蛋白质折叠方面取得实质性进展。我们在所提出的技术、获得所需的质粒、材料和检测方法方面拥有丰富的经验，并与 EM 和 ER 伴侣专家建立了合作关系。该研究促进了生物学、化学和物理学交叉领域的跨学科培训。