Calreticulin-mediated protein folding in health and disease

 描述(申请人提供)：内质网(ER)动态平衡的改变可由伴侣或其底物蛋白的突变引起。钙网蛋白是一种钙结合的内质网伴侣蛋白，对许多N-连接的糖蛋白的折叠和组装非常重要。在巨噬细胞和凋亡细胞的表面也发现了钙网蛋白，在那里它有助于细胞的吞噬。关于钙网蛋白依赖的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，仍有许多需要了解。此外，对钙网蛋白的细胞外功能知之甚少，包括与细胞-表面相互作用相关的机制，以及与钙网蛋白依赖的吞噬作用相关的机制。对于钙结合域改变的钙网蛋白突变体的功能丧失和获得也知之甚少，这种突变常见于骨髓增生性肿瘤(MPN)。其中一些知识差距将在本申请中得到解决。主要的假设是，ATP是钙网蛋白-底物相互作用的关键调节因子，不同的蛋白质和脂类识别模式是钙网蛋白细胞功能的中心。采用计算和实验相结合的方法，提出了一个计算钙网蛋白结合部位的模型。ATP结合被证明破坏了钙网蛋白与细胞单糖化的主要组织相容性复合体(MHC)I类分子的结合。还将检测三磷酸腺苷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)、其错误折叠突变体atz和低密度脂蛋白相关蛋白(lrp-1)。将研究钙网织蛋白诱导不溶性ATZ清除的分子机制，检验钙网织蛋白识别多肽与这一活性相关的模型。我们将研究底物和ER因素对核苷酸交换和钙网蛋白ATPase活性的影响。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的钙结合部位，也含有磷脂酰丝氨酸(PS)和凋亡细胞的结合部位。经常出现在MPN中的体细胞钙网织蛋白突变体改变了非酸性C末端。据预测，这些突变不仅会改变钙和PS的结合，以及依赖钙网蛋白的细胞吞噬作用，而且还会影响钙网蛋白的构象和伴侣活性，这一点将被研究。基于从这些研究中获得的知识，我们希望开发策略来促进蛋白质错误折叠障碍(如AAT缺乏症)中活性蛋白质的形成，并了解钙网织蛋白突变在癌症中的致病作用。

 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.