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

• 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
• 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

 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）：内质网（ER）稳态的改变可能是由分子伴侣或其底物蛋白的突变引起的。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，促进细胞吞噬作用。关于钙网蛋白依赖性蛋白质折叠的分子机制还有很多有待了解，包括调节内质网中底物结合和释放的因素。此外，人们对钙网蛋白的细胞外功能知之甚少，包括与钙网蛋白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。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，这种突变体常见于骨髓增生性肿瘤（MPN）中。本应用程序将解决其中一些知识差距。主要假设是 ATP 是钙网蛋白与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 结合显示会破坏钙网蛋白与细胞单糖基化主要组织相容性复合物 (MHC) I 类分子的结合稳定性。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，检查钙网蛋白识别多肽与该活性相关的模型。将研究底物和 ER 因子对核苷酸交换和钙网蛋白 ATP 酶活性的影响。初步数据表明，钙网蛋白的 C 端酸性结构域含有低亲和力钙结合位点，还含有磷脂酰丝氨酸 (PS) 和凋亡细胞的结合位点。 MPN 中经常出现的体细胞钙网蛋白突变体改变了非酸性 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，还会影响钙网蛋白的构象和伴侣活性，对此将进行研究。基于从这些研究中获得的知识，我们期望制定策略来增强蛋白质错误折叠疾病（例如 AAT 缺乏）中活性蛋白质的形成，并了解钙网蛋白突变对癌症的致病作用。