A Simple Secretion Assay for Assessing New and Existing Myocilin Variants.
A Simple Secretion Assay for Assessing New and Existing Myocilin Variants.
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DOI：
10.1080/02713683.2022.2047205
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发表时间：
2022-06
影响因子：
2
通讯作者：
中科院分区：
文献类型：
--
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缺乏足够的信息来适当地分类大量的肌球蛋白（MYOC）变体，以及它们参与原发性开角型青光眼，阻碍了它们的明确分类。大多数导致脑瘤的MYOC突变导致蛋白质不分泌和培养细胞中细胞内不溶性聚集体的形成。在本文中，我们产生了基于Gaussia内切酶的MYOC融合蛋白，以快速且灵敏地追踪MYOC变体的分泌，并将这些结果与更好地建立的MYOC蛋白质印迹分析进行比较。将具有不同程度的预测致病性的14种临床来源的MYOC变体转染到HEK-293 A细胞中，并通过荧光素酶测定或蛋白质印迹法进行分析。其中8种变体（G12 R、V53 A、T204 T、P254 L、T325 T、D380 H、D395_E396insDP和P481 S）之前未进行生化评估。除此之外，P254 L和D395_E396insDP表现出显著的分泌缺陷，这让人联想到引起结肠癌的突变。荧光素酶测定结果与14种变体中的13种（93%）的蛋白质印迹一致，表明强烈的一致性。这些结果表明，Gaussia荧光素酶测定可用作快速评估MYOC变体行为的补充或独立测定，我们预计这些结果将用于MYOC变体的治疗和重新分类。
A lack of sufficient information exists for appropriately classifying a large number of myocilin (MYOC) variants, and their involvement in primary open angle glaucoma, hindering their definitive classification. Most glaucoma-causing MYOC mutations result in protein non-secretion and intracellular insoluble aggregate formation in cultured cells. Herein, we generated a Gaussia luciferase-based MYOC fusion protein to quickly and sensitively track the secretion of MYOC variants and compared these results to the better-established MYOC assay of western blotting. Fourteen clinically-derived MYOC variants with varying degrees of predicted pathogenicity were transfected into HEK-293A cells and analyzed by either a luciferase assay or western blotting. Eight of the variants (G12R, V53A, T204T, P254L, T325T, D380H, D395_E396insDP, and P481S) had not been biochemically assessed previously. Off these, P254L and D395_E396insDP demonstrated significant secretion defects from reminiscent of glaucoma-causing mutations. The luciferase assay results agreed with western blotting for thirteen of the fourteen variants (93%), suggesting a strong concordance. These results suggest that the Gaussia luciferase assay may be used as a complementary or standalone assay for quickly assessing MYOC variant behavior and we anticipate that these results will be useful in MYOC variant curation and reclassification.
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影响因子：
4.2
作者：
Shimizu, S;Lichter, PR;Richards, JE
通讯作者：
Richards, JE
影响因子：
3.5
作者：
Zhou, ZH;Vollrath, D
通讯作者：
Vollrath, D
影响因子：
4.8
作者：
Hill, Shannon E.;Kwon, Michelle S.;Lieberman, Raquel L.
通讯作者：
Lieberman, Raquel L.
针对小梁网中的ER-自助系统来治疗青光眼。
影响因子：
3.4
作者：
Stothert AR;Fontaine SN;Sabbagh JJ;Dickey CA
通讯作者：
Dickey CA
一种新型的荧光素酶测定，用于敏感监测细胞培养中的肌动蛋白变体。
影响因子：
4.4
作者：
Zadoo S;Nguyen A;Zode G;Hulleman JD
通讯作者：
Hulleman JD