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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566677
• 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 In unpublished work, we have cloned, expressed and purified calnexin cycle components from yeast and a key ER sensor of unfolded proteins.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.

膜和分泌的蛋白质会获得翻译后修饰，并通过完成内质网（ER），高尔基体和秘密蔬菜的秘密途径折叠。为此，细胞已经进化出一组专业的链酮，酶和受体分子，这些链分子介导了蛋白质折叠和运输的多个步骤。我的研究介绍了ER中蛋白质折叠的两个方面：1）N-连锁糖蛋白的碳水化合物结构与伴侣募集的碳水化合物结构之间的联系，以及2）识别展开蛋白质的机制。这两个过程均由钙钙蛋白周期的伴侣进行。钙钙蛋白周期由伴侣组成，这些伴侣折叠糖蛋白和酶，这些糖蛋白和酶修饰附着的聚糖以反映蛋白质的折叠状态。该循环的功能是促进新合成的糖蛋白的有效折叠，并防止其过早出口ER。关于钙钙蛋白周期有许多未解决的问题：i）伴侣在较低的生物体中是否相似地发挥作用？ ii）钙钙蛋白周期如何区分折叠和展开的蛋白质？ iii）是否有关于如何识别出的蛋白质的一般代码？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，我们确定了肽基脯氨酰异构酶与钙网蛋白周期之间的新型关联。我们还确定了链酮钙网蛋白如何识别聚糖。在未发表的工作中，我们从酵母和展开的蛋白质的关键ER传感器中克隆，表达和纯化的钙网蛋白周期成分。在这里，我建议通过将结构生物学和体外功能测定法与重点关注两个目的的工作相结合，以继续这些研究：1）酵母中讲授链酮复合物的结构和功能研究。我们已经确定了酵母钙钙蛋白（CNE1P）的相互作用环，并表明它与酵母蛋白二硫化物异构酶（MPD1P）相互作用。我们将确定MPD1P上的结合表面，并使用该信息来指导复合物的共结晶。我们将进行功能评估，以测试我们的假设，即CNE1P•MPD1P与哺乳动物直系同源物相似。这项工作将使我们对钙钙蛋白周期的理解扩展到特征良好的酵母菌。 2）UDP-葡萄糖的研究：糖蛋白 - 葡萄糖基转移酶（UGGT）。该密钥ER酶专门为展开的蛋白质的N连接聚糖添加了葡萄糖居住。我们拥有广泛的初步数据，可用于从具有功能测定的多个物种纯化UGGT，以表明纯化的蛋白质是活跃的。自意向通知提交以来，我们通过电子显微镜（EM）取得了令人兴奋的进步。 UGGT的负染色3D重建可重复显示一个大型的中央空腔，我们假设这是催化部位。这将解释UGGT对展开的蛋白质的特异性。折叠域中的聚糖无法进入催化位点，而在未折叠的多肽链上的聚糖则能够进入腔室。疏水性保留的存在将有利于展开的蛋白质片段的结合，并进一步提高酶的选择性。我们将通过EM，X射线晶体学和UGGT的SAXS研究来检验这一假设。我的小组的位置很好，可以在理解ER中的蛋白质折叠方面取得重大进展。我们拥有提出的技术，访问所需的质粒，材料和测定法的经验，并与EM和ER伴侣的专家建立了合作。该研究促进了生物学，化学和物理学界面的跨学科培训。