Luminescent assay:ER dislocation inhibitors (RMI)

描述（由申请人提供）：在内质网（ER）中合成预定用于质膜或细胞外空间的蛋白质。 ER质量控制系统检查新合成的蛋白质，并确保只有正确折叠的蛋白质以及完全组装的复合物才允许离开ER。 由于基因突变而不能正确折叠的ER蛋白被蛋白酶体靶向在胞质溶胶中降解。 多肽从ER到胞质溶胶的提取被称为移位。 这些蛋白质包括可溶性以及膜分子。 与特定ER蛋白降解相关的疾病范围从引起法布里病和泰-萨二氏病的神经缺陷到肺部疾病如囊性纤维化和肺气肿。 病原体还利用破坏特定蛋白质来逃避免疫检测。 人巨细胞病毒（HCMV）编码的基因产物US 2和US 11介导免疫必需的膜蛋白MHC I类重链的破坏。 这些病毒基因产物以与错误折叠的ER蛋白类似的方式靶向MHC I类进行降解。 我们将建立一个生物发光测定人类细胞系，可用于高通量筛选，以确定ER错位的细胞渗透性抑制剂。 如果ER降解底物可以避开位错反应，则它们可以被重新定向到其适当的隔室，并可能防止疾病状态的发作。 由于被围困的错位/降解的拓扑多样性ER蛋白，我们将产生表达靶向错位/降解的可溶性或膜生物发光嵌合分子的人细胞系。 可溶性生物发光嵌合分子将由与α 1-抗胰蛋白酶的突变形式融合的增强型绿色荧光蛋白（EGFP）或荧光素酶组成。 HCMV US 11细胞中的嵌合EGFP/I类重链或荧光素酶/I类重链将用于代表膜蛋白的移位。 位错反应的抑制将导致生物发光嵌合分子的积累，并导致光能的增加。 这种光能的增加将为高通量筛选提供读数。 确定诱导能量放大的化学化合物将被考虑用于第二阶段分析，使用生物化学测定来验证其阻断位错的能力。

DESCRIPTION (provided by applicant): Proteins destined for the plasma membrane or extracellular space are synthesized in the endoplasmic reticulum (ER). The ER quality control system inspects newly synthesized proteins and ensures that only correctly folded proteins as well as fully assembled complexes are allowed to leave the ER. ER proteins that do not fold correctly due to genetic mutations are targeted for degradation in the cytosol by the proteasome. The extraction of the polypeptide from the ER to the cytosol is referred to as dislocation. These proteins include soluble as well as membrane molecules. Diseases associated with the degradation of specific ER proteins range from neurological defects that cause Fabri disease and Tay-Sachs to lung diseases such as cystic fibrosis and emphysema. Pathogens also utilize the destruction of specific proteins to evade immune detection. The human cytomegalovirus (HCMV) encoded gene products US2 and US11 mediate the destruction of the immunologically essential membrane protein MHC class I heavy chain. These viral gene products target MHC class I for degradation in a similar manner as misfolded ER proteins. We will establish a bioluminescent assay in human cell lines that can be used in a high-throughput screen to identify cell permeable inhibitors of ER dislocation. If ER degradation substrates can elude the dislocation reaction, they may be re-directed to their proper compartment and possibly prevent the onset of the disease state. Due to the topologically diverse ER proteins besieged for dislocation/degradation, we will generate human cell lines that express either a soluble or membrane bioluminescent chimeric molecule that is targeted for dislocation/degradation. The soluble bioluminescent chimeric molecule will be comprised of either enhanced green fluorescent protein (EGFP) or luciferase fused to a mutant form of a1-antitrypsin. Chimeric EGFP/class heavy chain or luciferase/class I heavy chain in HCMV US11 cells will be utilized to represent the dislocation of membrane proteins. Inhibition of the dislocation reaction would bring about an accumulation of bioluminescent chimeric molecules and cause an augmentation in light energy. This increase in light energy will provide the readout for the high throughput screen. Chemical compounds identified to induce an amplification in energy will be considered for second phase analysis using biochemical assays to validate their ability to block dislocation.