Identification of Inhibitors that Stabilize ER Degradation Substrates

稳定 ER 降解底物的抑制剂的鉴定

• 批准号: 8286199
• 负责人: Domenico Tortorella
• 金额: $ 4.24万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-20 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8286199
• 关键词: Biological Assay; Carrier Proteins; Cells; Chimera organism; Cholera; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytomegalovirus; Cytosol; Cytotoxic T-Lymphocytes; Destinations; Detection; Disease; Dislocations; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Ensure; Fluorescence; Genetic; Half-Life; Hematological Disease; Hereditary Disease; Hour; Immune; Immune system; Kinetics; Libraries; Lung diseases; Major Histocompatibility Complex; Mammalian Cell; Mediating; Membrane Proteins; Metabolism; Molecular Bank; Molecular Conformation; Mutation; Neuronal Ceroid-Lipofuscinosis; Pharmaceutical Preparations; Process; Production; Protein C Deficiency; Protein C Inhibitor; Proteins; Pulmonary Emphysema; Quality Control; Reagent; Resources; Ricin; Screening procedure; System; T-Cell Receptor; Toxin; Variant; Viral; Virus; base; eeyarestatin I; high throughput screening; human disease; immune clearance; inhibitor/antagonist; multicatalytic endopeptidase complex; mutant; nervous system disorder; next generation; novel; pathogen; prevent; protein degradation; protein misfolding; small molecule libraries; trafficking

DESCRIPTION (provided by applicant): Newly synthesized endoplasmic reticulum (ER) proteins are subjected to inspection by the ER quality control machinery to ensure that only properly folded molecules can traffic to their final destination. ER proteins that cannot reach their native state due to genetic defects are exported out of the ER and into the cytosol by a multi- step process referred to as dislocation, followed by proteasome degradation generally referred to as ER-associated degradation (ERAD). Human diseases whose genetic defects prevent the protein from reaching its native conformation include pulmonary diseases (e.g. emphysema and cystic fibrosis), blood disorders (e.g. protein C deficiency), and neurological disorders (e.g. neuronal ceroid lipofuscinoses and Fabri disease). In addition, toxins such as cholera and ricin toxin have exploited the transport of proteins from the ER to cytosol to gain access to the cytosol and alter cellular metabolism. Strikingly, viruses have evolved to utilize the ERAD pathway to evade immune detection and persist within the host. Human cytomegalovirus (HCMV) unique short (US)2 and US11 gene products have co-opted ERAD to mediate the destruction of the immunological protein major histocompatibility complex (MHC) class I heavy chain to limit viral clearance by cytotoxic T cells. In fact, how proteins are extracted from the ER in mammalian cells was initially characterized using HCMV US2- and US11-expressing cells and this cell system continues to identify key ERAD components. An important feature of US2 and US11-mediated class I destruction is the fast kinetics (t1/2 ~5 min) of class I degradation. The short half-life of class I heavy chains in US2 and US11 cells was adapted into a high-throughput screen using a class I heavy chain chimera consisting of enhanced green fluorescence protein and class I heavy chain. Two compounds referred to as eeyarestatin I and II were identified to stabilize class I heavy chains as well as other misfolded proteins, T cell receptor 1 chain and an 11-antitrypsin variant. The screening of larger and more diverse chemical libraries would allow for the discovery of additional compounds that stabilize ER degradation substrates. These inhibitors would be effective agents against genetic disorders caused by misfolded ER proteins and pathogens that utilize the ERAD pathway.

描述（由申请方提供）：新合成的内质网（ER）蛋白由ER质量控制机构进行检查，以确保只有正确折叠的分子才能运输到其最终目的地。由于遗传缺陷而不能达到其天然状态的ER蛋白通过称为错位的多步骤过程从ER输出并进入细胞溶质，随后是通常称为ER相关降解（ERAD）的蛋白酶体降解。其遗传缺陷阻止蛋白质达到其天然构象的人类疾病包括肺部疾病（例如肺气肿和囊性纤维化）、血液病症（例如蛋白C缺乏症）和神经病症（例如神经元蜡样脂褐质沉积症和法布里病）。此外，毒素如霍乱和蓖麻毒素已经利用蛋白质从ER到胞质溶胶的运输以进入胞质溶胶并改变细胞代谢。引人注目的是，病毒已经进化到利用ERAD途径来逃避免疫检测并在宿主内持续存在。人巨细胞病毒（HCMV）独特的短（US）2和US 11基因产物已增选ERAD介导免疫蛋白主要组织相容性复合体（MHC）I类重链的破坏，以限制细胞毒性T细胞对病毒的清除。事实上，最初使用表达HCMV US 2和US 11的细胞来表征如何从哺乳动物细胞的ER中提取蛋白质，并且该细胞系统继续识别关键的ERAD成分。US 2和US 11介导的I类破坏的一个重要特征是I类降解的快速动力学（t1/2 ~5 min）。使用由增强的绿色荧光蛋白和I类重链组成的I类重链嵌合体，将US 2和US 11细胞中I类重链的短半衰期调整为高通量筛选。两种化合物被称为eeyarestatin I和II被鉴定为稳定I类重链以及其他错误折叠的蛋白质，T细胞受体1链和11-抗胰蛋白酶变体。筛选更大和更多样化的化学文库将允许发现稳定ER降解底物的其他化合物。这些抑制剂将是有效的药物，对遗传疾病引起的错误折叠的ER蛋白和病原体，利用ERAD途径。