Chemical chaperone therapy: Luciferase assay for screening of β-galactosidase mutations
Chemical chaperone therapy: Luciferase assay for screening of β-galactosidase mutations
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DOI：
10.1016/j.ymgme.2010.08.012
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发表时间：
2010-12-01
影响因子：
3.8
通讯作者：
Nanba, Eiji
中科院分区：
文献类型：
--
作者：
Li, Linjing;Higaki, Katsumi;Nanba, Eiji
β-半乳糖苷沉积症是一组基于编码溶酶体酸性 β-半乳糖苷酶 (β-gal) 的 GLB1 基因异质突变的疾病。 β-半乳糖酶活性的降低导致体细胞中底物的逐渐积累，特别是在神经元中，导致严重的神经元功能障碍。我们之前曾报道过，N-辛基-4-表-β-缬烯胺 (NOEV) 是一种化学伴侣化合物，可以稳定各种突变的人类 β-gal 蛋白，并增加人类患者培养的成纤维细胞中的残留酶活性。这些数据证明化学伴侣疗法对错义β-gal患者具有潜在的治疗益处。这种效应是突变特异性的。在这项研究中，我们建立了一种基于荧光素酶的灵敏测定法，用于测量突变型人 β-gal 的伴侣效应。将甲藻荧光素酶 (Dluc) cDNA 引入人 β-gal 的 C 末端。当表达 Dluc 标记的人 R201C β-gal 的 COS7 细胞用 NOEV 处理时，突变型 β-gal 活性显着增加。在 NH4Cl 存在的情况下，培养基中的荧光素酶水平与细胞裂解物中的酶活性同时增加。我们还发现蛋白酶体抑制剂增强了 NOEV 的伴侣效应。这些结果表明，基于荧光素酶的测定是筛选和评估人类β-gal突变体伴侣效应的可靠且方便的方法，并且它将成为在未来研究中寻找新型伴侣化合物的有用工具。 (C) 2010 Elsevier Inc. 保留所有权利。
beta-Galactosidosis is a group of disorder based on heterogeneous mutations of GLB1 gene coding for the lysosomal acid beta-galactosidase (beta-gal). A decrease of the beta-gal enzyme activity results in progressive accumulation of substrates in somatic cells, particularly in neurons, leading to severe neuronal dysfunction. We have previously reported that N-octyl-4-epi-beta-valienamine (NOEV), a chemical chaperone compound, stabilized various mutant human beta-gal proteins and increased residual enzyme activities in cultured fibroblasts from human patients. These data proved a potential therapeutic benefit of chemical chaperone therapy for patients with missense beta-gal. This effect is mutation specific. In this study, we have established a sensitive luciferase-based assay for measuring chaperone effect on mutant human beta-gal. A dinoflagellate luciferase (Dluc) cDNA was introduced to the C-terminus of human beta-gal. When COS7 cells expressing the Dluc-tagged human R201C beta-gal was treated with NOEV, there happened a remarkable increase of the mutant beta-gal activity. In the presence of NH4Cl, luciferase level in the medium increased in parallel with the enzyme activity in cell lysates. We also found that proteasome inhibitors enhance chaperone effect of NOEV. These results demonstrate that the luciferase-based assay is a reliable and convenient method for screening and evaluation of chaperone effects on human beta-gal mutants, and that it will be a useful tool for finding novel chaperone compounds in the future study. (C) 2010 Elsevier Inc. All rights reserved.