Cell-Penetrable and in vivo activatable mini-chaperones: A potential therapeutic strategy for stabilizing protein misfolding in disease states.

摘要 蛋白质错误折叠及其致病后果已成为人类疾病的重要因素。 眼部各种蛋白质构象疾病，包括白内障、视网膜色素变性、青光眼、黄斑 变性、晶格状和颗粒状角膜营养不良等，遵循常见的结构和病理 路径。防止蛋白质聚集对于生物功能的保存至关重要。在……下面 作为预防蛋白质错误折叠和随之而来的细胞功能障碍和死亡的一线疗法的发展 有各种分子伴侣，包括小分子和蛋白质和多肽伴侣 选择性地结合并稳定靶蛋白。最近，一种迷你伴侣(DFVIFLDVKHFSPEDLTVK)，a 从我们实验室鉴定的αA-晶体蛋白伴侣位点衍生的多肽被证明稳定了各种 错误折叠的蛋白质并缓解相关的病理，表明迷你伴侣可能是 被开发为对抗涉及蛋白质聚集、氧化应激、 细胞凋亡和炎症。我们建议提高多肽伴侣的体内治疗效果。 并揭示其作为一种通用伴侣用于蛋白质构象疾病的潜力。我们假设 (1)通过使用D-氨基酸(TO)可以提高伴侣肽的治疗效果 增加半衰期)在其合成和细胞穿透序列的结合期间(用于快速摄取)和(2) 多肽伴侣的特异性可以通过活性物质的水解和释放来增强 仅在预定的细胞和组织中通过应激特定的蛋白酶的作用而产生的伴侣多肽。测试我们的 假设，我们将合成并表征穿透细胞和体内可激活的迷你伴侣 (CPIAMCs)，并测定其在原代晶状体上皮细胞、ARPE-19细胞和n27神经细胞中的伴侣活性 细胞，以评估其作为治疗性多肽伴侣的潜力。 本项目的具体目标是-目标1(A)合成和表征CPIAMC多肽(S)，将 包括描述CPIAMCs的细胞摄取、毒性和体内激活；和(B)调查 CPIAMCs保护细胞免受氧化应激的功效。目标2：(A)确定CPIAMCs对以下方面的影响 两种致病突变体myoclin(Y437H)(与青光眼有关)的分泌和积累 TM-3细胞和转化生长因子β诱导蛋白(R124H)(与颗粒状角膜有关) 营养不良)和(B)评估CPIAMCs抑制未折叠蛋白的能力 肌球蛋白Y437H或转化生长因子β诱导突变蛋白表达细胞的反应及相关毒性 R124H。分子伴侣设计的新策略将导致开发新的工具 蛋白质聚集性疾病的治疗药物，最终改善人们的生活质量 患有这种疾病，每年节省数百亿美元的医疗费用 疾病。

Abstract Protein misfolding and its pathogenic consequences have emerged as important factors in human disease. Diverse protein conformational diseases of the eye, including cataract, retinitis pigmentosa, glaucoma, macular degeneration, lattice and granular corneal dystrophies, etc., follow a common structural and pathological pathway. Prevention of protein aggregation is crucial for the preservation of biological function. Under development as frontline therapies to prevent protein misfolding and the ensuing cellular dysfunction and death are various molecular chaperones, including small molecules and protein and peptide chaperones that selectively bind to and stabilize the target proteins. Recently, a mini-chaperone (DFVIFLDVKHFSPEDLTVK), a peptide derived from the αA-crystallin chaperone site and identified in our lab, was shown to stabilize various misfolded proteins and alleviate the associated pathology, suggesting that the mini-chaperone could be developed as a “universal chaperone” against diseases involving protein aggregation, oxidative stress, apoptosis, and inflammation. We propose to improve the in vivo therapeutic efficacy of the peptide chaperone and uncover its potential as a universal chaperone for use in protein conformational diseases. We hypothesize that (1) the therapeutic efficacy of the chaperone peptide can be improved by use of D-amino acids (to increase half-life) during its synthesis and conjugation of a cell-penetrating sequence (for rapid uptake) and (2) the specificity of the peptide chaperone can be enhanced with the hydrolysis and release of the active chaperone peptide by the action of stress-specific proteases only in intended cells and tissues. To test our hypothesis, we will synthesize and characterize the cell-penetrating and in vivo activatable mini-chaperones (CPIAMCs) and determine its chaperone activity in primary lens epithelial cells, ARPE-19 cells, and N27 neural cells, to evaluate its potential as therapeutic peptide chaperone. The Specific Aims of this project are — Aim 1 (a) Synthesis and characterization of CPIAMC peptide(s) that will encompass delineating the cellular uptake, toxicity and in vivo activation of CPIAMCs; and (b) investigation of the CPIAMCs efficacy to protect cells from oxidative stress. AIM 2: (a) Determine the effects of CPIAMCs on the secretion and accumulation of two disease-causing mutants, myocilin (Y437H) (implicated in glaucoma) in TM-3 cells and transforming growth factor beta-induced protein (R124H) (implicated in granular corneal dystrophy) in corneal fibroblasts and (b) evaluate the ability of CPIAMCs to suppress unfolded protein response and associated toxicity in cells expressing the myocilin Y437H or TGFβ –induced mutant protein R124H. The novel strategy for the design of molecular chaperones will lead to new tools for the development of therapeutic agents for protein aggregation diseases, ultimately improving the quality of life of persons suffering from such diseases and saving of tens of billions of dollars each year in healthcare costs for these diseases.