Calreticulin-mediated protein folding in health and disease

健康和疾病中钙网蛋白介导的蛋白质折叠

基本信息

批准号：
9238654
负责人：
MALINI RAGHAVAN
金额：
$ 38.75万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-10 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9238654
关键词：
ATP phosphohydrolase Address Affect Affinity Apoptotic Area Binding Binding Sites Biological Response Modifiers Biology C-terminal Calcium Calcium Binding Calcium-Binding Domain Cell Count Cell Surface Receptors Cell physiology Cell secretion Cell surface Cells Complex Data Development Disease Endoplasmic Reticulum Frequencies Functional disorder Future Glycoproteins Health Hereditary Disease Homeostasis Immune response In Vitro Inflammatory Response Knowledge LDL-Receptor Related Proteins Link Lipids Lysosomal Storage Diseases Major Histocompatibility Complex Malignant Neoplasms Measures Mediating Mediator of activation protein Metabolic Clearance Rate Mission Modeling Molecular Molecular Chaperones Molecular Conformation Mutate Mutation Nature Neoplasms Nucleotides Pathogenicity Phagocytes Phagocytosis Phosphatidylserines Phospholipids Prognostic Marker Protein Secretion Protein-Folding Disease Proteins Proteomics Quality Control Regulation Research Role Solubility Substrate Interaction Surface System United States National Institutes of Health Variant Work alpha 1-Antitrypsin alpha 1-Antitrypsin Deficiency base burden of illness calreticulin cancer therapy disability extracellular gain of function human disease immune function in vivo insight macrophage misfolded protein mutant novel polypeptide protein folding protein misfolding proteostasis public health relevance

项目摘要

DESCRIPTION (provided by applicant): Alterations of endoplasmic reticulum (ER) homeostasis can result from mutations of chaperones or of their substrate proteins. Calreticulin is a calcium-binding ER chaperone that is important for the folding and assembly of many N-linked glycoproteins. Calreticulin is also found on the surface of macrophages and apoptotic cells, where it facilitates cellular phagocytosis. Much remains to be understood about the molecular mechanisms of calreticulin-dependent protein folding, including factors that regulate substrate binding and release in the ER. Furthermore, the extracellular functions of calreticulin are poorly understood, including the mechanisms relevant to cell-surface interactions of calreticulin, and to calreticulin-dependent phagocytosis. There is also little knowledge about the loss and gain of function of calreticulin mutants with altered calcium- binding domains that are frequently found in myleoproliferative neoplasms (MPN). Some of these gaps in knowledge will be addressed in this application. The main hypotheses are that ATP is a key regulator of calreticulin-substrate interactions and that distinct modes of protein and lipid recognition are central to the cellular functions of calreticulin. Using computational and experimental approaches, a model for the ATP binding site of calreticulin is presented. ATP binding is shown to destabilize calreticulin binding to cellular monoglucosylated major histocompatibility complex (MHC) class I molecules. The effect of ATP binding deficient mutants on the maturation of other substrates will be examined, including α1-antitrypsin (AAT), its misfolded variant ATZ, and the low-density lipoprotein-related protein (LRP-1). The molecular mechanisms by which calreticulin induces the clearance of insoluble ATZ will be studied, examining the model that polypeptide recognition by calreticulin is relevant to this activity. The influences of substrates and ER factos upon nucleotide exchange and upon the ATPase activity of calreticulin will be studied. Preliminary data indicate that the C-terminal acidic domain of calreticulin, which contains low affinity calcium-binding sites, also contains binding sites for phosphatidylserine (PS) and apoptotic cells. Somatic calreticulin mutants that are frequently present in MPN have altered non-acidic C-termini. These mutations are predicted to not only alter calcium and PS binding, and calreticulin-dependent cellular phagocytosis, but also affect the conformation and chaperone activity of calreticulin, which will be studied. Based on the knowledge gained from these studies, we expect to develop strategies to enhance the formation of active proteins in protein misfolding disorders such as AAT deficiency, and to understand the pathogenic effects of calreticulin mutations in cancer.

描述（由申请人提供）：伴侣或其底物蛋白的突变可能导致内质网 (ER) 稳态的改变。钙网蛋白是一种钙结合 ER 伴侣，对于许多 N 连接糖蛋白的折叠和组装非常重要。钙网蛋白也存在于巨噬细胞和凋亡细胞的表面，它可以促进细胞的吞噬作用。人们对钙网蛋白依赖性蛋白质折叠的分子机制，包括调节内质网中底物结合和释放的因素了解甚少。此外，对钙网蛋白的细胞外功能知之甚少，包括与钙网蛋白的细胞表面相互作用相关的机制，以及与钙网蛋白的细胞表面相互作用相关的机制。钙网蛋白依赖性吞噬作用对于具有改变的钙结合结构域的钙网蛋白突变体的功能丧失和获得也知之甚少，这种突变体经常在骨髓增殖性细胞中发现。本申请将解决其中一些知识空白，主要假设是 ATP 是钙网蛋白-底物相互作用的关键调节剂，并且蛋白质和脂质识别的不同模式是钙网蛋白的细胞功能的核心。使用计算和实验方法，提出了钙网蛋白 ATP 结合位点的模型，该模型显示钙网蛋白与细胞单糖基化主要组织相容性复合物的结合不稳定。 (MHC) I 类分子的 ATP 结合缺陷突变体对其他底物成熟的影响将被检查，包括 α1-抗胰蛋白酶 (AAT)、其错误折叠变体 ATZ 和低密度脂蛋白相关蛋白 (LRP-1)。）将研究钙网蛋白诱导不溶性 ATZ 清除的分子机制，检查钙网蛋白的多肽识别与该活性相关的模型。初步数据表明，钙网蛋白的 C 末端酸性结构域含有低亲和力钙结合位点，也含有磷脂酰丝氨酸 (PS) 和细胞凋亡的结合位点。 MPN 中经常存在的体细胞钙网蛋白突变体已经改变了非酸性 C 末端，预计这些突变不仅会改变钙。和 PS 结合以及钙网蛋白依赖性细胞吞噬作用，而且还会影响钙网蛋白的构象和伴侣活性，基于从这些研究中获得的知识，我们期望开发出增强蛋白质中活性蛋白形成的策略。错误折叠疾病如 AAT 缺乏，并了解钙网蛋白突变对癌症的致病作用。