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（由适用提供）：内质网（ER）稳态的改变可能是由链酮或其底物蛋白的突变引起的。钙网蛋白是一种钙结合ER链酮，对于许多N连接糖蛋白的折叠和组装很重要。钙网蛋白还在巨噬细胞和凋亡细胞的表面上发现，在那里它促进了细胞吞噬作用。关于钙网蛋白依赖性蛋白折叠的分子机制，还有许多待理解的知识，包括调节底物结合并释放在ER中的因素。此外，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以及钙网蛋白依赖性吞噬作用相关的机制。关于钙蛋白结合域改变的钙网蛋白突变体功能的损失和功能的知识也很少，而钙结合结构域经常在肌毛增生性肿瘤（MPN）中发现。知识中的一些差距将在本应用程序中解决。主要的假设是ATP是钙网蛋白 - 底物相互作用的关键调节剂，而不同的蛋白质和脂质识别模式对于钙网蛋白的细胞功能至关重要。使用计算和实验方法，提出了钙网蛋白ATP结合位点的模型。 ATP结合显示可破坏钙网蛋白与细胞单糖基化的主要组织相容性复合物（MHC）I类分子的结合。将检查ATP结合缺陷突变体对其他底物成熟的影响，包括α1-抗胰蛋白酶（AAT），其错误折叠的变体ATZ和低密度脂蛋白相关蛋白（LRP-1）。钙网蛋白诱导不溶性ATZ清除率的分子机制将进行研究，研究模型，即通过钙网蛋白识别多肽与该活性有关。核交换时底物和ER事实的影响以及钙网蛋白的ATPase活性的影响将是研究的。初步数据表明，钙网蛋白的C末端酸性结构域，其中含有低亲和力钙结合位点，还包含用于磷脂酰丝氨酸（PS）和凋亡细胞的结合位点。 MPN中经常存在的体细胞钙蛋白突变体已改变了非酸性C-末端。预测这些突变不仅会改变钙和PS结合，并依赖钙网蛋白依赖性细胞吞噬作用，而且还会影响钙网蛋白的会议和链酮活性，这将被研究。基于这些研究所获得的知识，我们希望制定策略，以增强蛋白质错误折叠疾病（例如AAT缺乏症）中活性蛋白的形成，并了解癌症中钙网蛋白突变的致病作用。