Luminescent assay:ER dislocation inhibitors (RMI)

描述(申请人提供)：进入质膜或细胞外空间的蛋白质是在内质网(ER)中合成的。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和完全组装的复合体才能离开ER。由于基因突变而不能正确折叠的ER蛋白是蛋白酶体在胞浆中降解的目标。将内质网中的多肽提取到胞浆中称为错位。这些蛋白质包括可溶性分子和膜分子。与特定ER蛋白降解相关的疾病从导致Fabri病和Tay-Sachs的神经缺陷到囊性纤维化和肺气肿等肺部疾病。病原体还利用对特定蛋白质的破坏来逃避免疫检测。人巨细胞病毒(HCMV)编码基因产物US2和US11介导了免疫必需的膜蛋白MHC I类重链的破坏。这些病毒基因产品以MHC I类为靶点进行降解，其方式与错误折叠的ER蛋白类似。我们将在人类细胞系中建立一种生物发光分析，可用于高通量筛选，以确定细胞通透性的内质网错位抑制物。如果内质网降解底物能够避开错位反应，它们可能被重新定向到适当的隔室，并可能防止疾病状态的发生。由于拓扑上不同的内质网蛋白被错位/降解包围，我们将产生表达可溶性或膜生物发光嵌合分子的人类细胞系，该嵌合分子是错位/降解的靶标。可溶的生物发光嵌合分子将由增强型绿色荧光蛋白(EGFP)或融合到突变形式的A1-抗胰蛋白酶的荧光素酶组成。人巨细胞病毒US11细胞中嵌合的EGFP/类重链或荧光素酶/I类重链将被用来代表膜蛋白的错位。抑制位错反应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屏幕提供读数。被确定为诱导能量放大的化合物将被考虑用于第二阶段的分析，使用生化分析来验证它们阻止位错的能力。

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.