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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587956
• 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 In unpublished work, we have cloned, expressed and purified calnexin cycle components from yeast and a key ER sensor of unfolded proteins.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。