Calreticulin's functions in the adaptive immune response

钙网蛋白是一种内质网（ER）伴侣蛋白，它促进钙网蛋白的折叠和组装。 糖蛋白，包括主要组织相容性复合体（MHC）I类分子。钙网蛋白也具有 将外源性抗原引导到MHC I类抗原呈递途径的能力，这是一种现象， 称为交叉呈现。作为一种凝集素，钙网蛋白与单葡萄糖基化核心聚糖相互作用， 糖蛋白在一定条件下，钙网蛋白能够结合底物的多肽组分。 钙耗竭和热处理暴露钙网蛋白的多肽结合位点， 在体外和细胞中与多肽底物结合。这些处理也诱导钙网蛋白二聚化 和低聚反应。钙网蛋白二聚体的形成还由其他类型的ER应激诱导， 包括病毒感染和衣霉素治疗。我们假设这些构象转变 多肽结合特性对钙网蛋白的蛋白质折叠和交叉启动功能非常重要 在细胞中。第一个具体的目的是探讨钙网蛋白在MHC I类分子中多肽结合的作用。 在细胞中折叠和组装。我们提出了部分蛋白水解和基于质谱的方法， 鉴定通过钙耗竭动员钙网蛋白亚结构域。保守疏水残基 钙网蛋白，预计是表面暴露，将突变为丙氨酸。突变体表现出 在体外与MHC I类重链的多肽组分相互作用的缺陷，以及其他 具有结合寡糖底物缺陷的突变体，将在钙网蛋白缺陷细胞中表达， 并评估促进MHC I类折叠和组装的能力。这些研究将 使我们能够完善我们的工作模型的钙网蛋白底物相互作用周期，其中交替 提出了与底物的寡糖和多肽组分的相互作用。我们将尝试 使钙网蛋白的截短形式结晶，所述截短形式具有增强的结合多肽底物的能力，和 也使钙网蛋白与鸡IgY片段的复合物结晶。第二个具体目标将探讨 钙网蛋白介导的交叉呈递机制。钙网蛋白的细胞内运输 将评估交叉呈递过程中钙网蛋白相关肽，以研究 一条内体-高尔基体网络-内质网运输路线。交叉呈递对钙网蛋白的要求 还将评估与凋亡细胞相关的抗原的活性。最后，研究了ER应激对细胞周期的影响。 钙网蛋白运输、细胞表面表达以及与抗原呈递细胞上的受体的相互作用将 进行评估。了解钙网蛋白功能的分子机制，阐明钙网蛋白的作用机制， 增强钙网蛋白的T细胞引发活性的条件将促进更有效的疫苗设计。

Calreticulin is an endoplasmic reticulum (ER) chaperone that promotes folding and assembly of glycoproteins, including major histocompatibility complex (MHC) class I molecules. Calreticulin also has the capacity to direct exogenous antigens onto the MHCclass I antigen presentation pathway, a phenomenon called cross-presentation. As a lectin, Calreticulin interacts with monoglucosylated core glycans on glycoproteins. Under certain conditions, Calreticulin is able to bind polypeptide components of substrates. Calcium depletion and heat-treatment expose calreticulin's polypeptide binding site and enhance Calreticulin binding to polypeptide substrates in vitro and in cells. These treatments also induce Calreticulin dimerization and oligomerization. The formation of Calreticulin dimers is additionally induced by other types of ER stress, including virus infection and tunicamycin treatment. It is our hypothesis that these conformational transitions and polypeptide-binding properties are important for calreticulin's protein folding and cross-priming functions in cells. The first specific aim explores the role of polypeptide binding by Calreticulin during MHC class I folding and assembly in cells. We propose partial proteolysis and mass spectrometry-based approaches to identify Calreticulin sub-domains that are mobilized by calcium depletion. Conserved hydrophobic residues of Calreticulin, that are predicted to be surface-exposed, will be mutated to alanines. Mutants that display defects in interactions with polypeptide components of MHC class I heavy chains in vitro, as well as other mutants with defects in binding oligosaccharide substrates, will be expressed in calreticulin-deficient cells, and assessed for the ability to facilitate MHC class I folding and assembly. Together, these studies will allow us to refine our working model for the calreticulin-substrate interaction cycle, in which alternating interactions with oligosaccharide and polypeptide components of substrates are proposed. We will attempt to crystallize truncated versions of Calreticulin that have enhanced ability to bind polypeptide substrates, and also crystallize Calreticulin complexes with chicken IgY fragments. The second specific aim will explore mechanisms of calreticulin-mediated cross-presentation. Intracellular trafficking of Calreticulinand calreticulin-associated peptides during cross-presentation will be assessed, to investigate the hypothesis of an endosome-trans Golgi network-ER trafficking route. The requirement for Calreticulin for cross-presentation of antigens associated with apoptotic cells will also be assessed. Finally, the effects of ER stress on Calreticulin trafficking, cell surface expression, and interactions with receptors on antigen presenting cells will be assessed. Understanding the molecular mechanisms of calreticulin's functions, and elucidation of conditions that enhance calreticulin's T cell priming activities, will facilitate more effective design of vaccines.