Calreticulin-mediated protein folding in health and disease

摘要 钙网蛋白(CRT)是一种钙结合内质网(ER)伴侣蛋白，对折叠非常重要 许多N-连接的糖蛋白。最为人所知的是它在主要组织相容性的组装中的功能。 复合体(MHC)I类(MHC-I)分子，呈递多肽抗原给CD8+T细胞以保护 对癌症和感染具有免疫力。最近的研究表明，体细胞移码突变改变了 CRT C末端是骨髓增生性肿瘤(MPN)中的一类驱动突变。52个碱基对 缺失突变体CRTDel52和5碱基对插入突变体CRTIns5是必需的反复突变 血小板增多症(ET)、原发性骨髓纤维化(PMF)和ET后骨髓纤维化(MF)。这是我们的整体 假设CRT的C-末端结构域的序列和电荷的变化是由 突变使MPN中的巨核细胞转化和免疫系统逃避。虽然 巨核细胞的致癌转化需要突变的CRT和血小板生成素 受体/骨髓增殖性白血病蛋白(MPL)--突变CRT介导的分子机制 MPL的结构性激活尚不清楚。已知CRT对内质网钙存储和细胞 钙信号，但MPN突变是如何改变这些过程的还不清楚。人类 MHC-I基因座具有高度多态现象。虽然已知MPN连接的突变CRT是无效的 被整合到MHC-I多肽负载复合体(PLC)中，目前尚不清楚MPN CRT突变体是否有 不同对不同MHC-I同种异型组装的影响。在此建议进行研究以解决这些问题 知识上的差距。我们提出的证据表明CRTDel52的二硫键连接二聚体对MPL很重要 激活。CRTDel52二聚体及其与MPL的络合物的性质将通过结构和 诱变研究。CRT缺乏、单倍体功能不全和突变对细胞内钙离子的影响 将检查信令。基于细胞表面CRT可变依赖关系的知识 MHC-I同种异型的表达，我们将检查MPN CRT突变是否损害MHC-I类组装 一些同种异型，但改变组装和肽显示的一些其他MHC-I同种类型。这样的改变可能会 可用于MPN的免疫治疗靶向。最后，突变的CRT表达也可在 MPN患者的血单核细胞，导致抗原提呈细胞(APC)钙变化的预测 信号和吞噬作用，这将被检查。综上所述，这些研究将推动我们的 了解普遍存在的内质网伴侣的突变如何驱动细胞转化，并影响 抗原呈递和吞噬的基本免疫学过程。

Abstract Calreticulin (CRT) is a calcium-binding endoplasmic reticulum (ER) chaperone that is important for the folding of many N-linked glycoproteins. It is best known for its function in the assembly of major histocompatibility complex (MHC) class I (MHC-I) molecules, which present peptide antigens to CD8+ T cells for protective immunity against cancers and infections. Recent studies show that somatic frameshift mutations that alter the C-terminus of CRT are one class of driver mutations in myeloproliferative neoplasms (MPN). A 52 base-pair deletion mutant CRTDel52 and a 5 base-pair insertion mutant CRTIns5 are recurrent mutations in essential thrombocythemia (ET), primary myelofibrosis (PMF) and post-ET myelofibrosis (MF). It is our overall hypothesis that the changes to the sequence and charge of the C-terminal domains of CRT induced by the mutations enable both megakaryocyte transformation and immune system evasion in MPN. Although oncogenic transformation in megakaryocytes requires both mutated CRT and the thrombopoietin receptor/myeloproliferative leukemia protein (Mpl), the molecular mechanisms of mutant CRT-mediated constitutive activation of Mpl are unknown. CRT is known to be important for ER calcium storage and cellular calcium signaling, but precisely how MPN mutations alter these processes is not understood. The human MHC-I locus is highly polymorphic. While it is known that MPN-linked mutant CRTs are ineffectively incorporated into the MHC-I peptide-loading complex (PLC), it is unknown whether MPN CRT mutants have differential influences on the assembly of various MHC-I allotypes. Studies are proposed here to address these gaps in knowledge. We present evidence that disulfide-linked dimers of CRTDel52 are important for Mpl activation. The nature of CRTDel52 dimers and their complexes with Mpl will be elucidated using structural and mutagenesis studies. The effects of CRT deficiency, haploinsufficiency and mutation upon cellular calcium signaling will be examined. Based on the knowledge of variable dependencies on CRT for cell-surface expression of MHC-I allotypes, we will examine whether MPN CRT mutants impair MHC class I assembly for some allotypes, but alter assembly and peptide display for some other MHC-I allotypes. Such alterations could be exploited for immunotherapeutic targeting of MPN. Finally, mutant CRT expression is also detectable in blood monocytes of MPN patients, leading to predictions of alterations to antigen presenting cell (APC) calcium signaling and phagocytosis, which will be examined. Taken together, these studies will further our understanding of how mutations of a ubiquitous ER chaperone drive cell transformation, and influence the fundamental immunological processes of antigen presentation and phagocytosis.