A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture.
A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture.
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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。
DOI：
10.1167/iovs.15-18789
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发表时间：
2016-04-01
影响因子：
4.4
通讯作者：
Hulleman JD
中科院分区：
文献类型：
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作者：
Zadoo S;Nguyen A;Zode G;Hulleman JD
原发性开角型青光眼相关的肌球蛋白（MYOC）突变导致蛋白质“不分泌”，使得分泌的MYOC难以使用常规技术检测。这项研究的重点是开发和使用一种可以快速简便地检测突变MYOC分泌的测定法。我们将Gaussia荧光素酶（eGLuc 2）与MYOC变体融合，并在HEK-293 T和NTM-5细胞中表达构建体。通过蛋白质印迹法评估MYOC eGLuc 2变体的分泌水平和细胞内水平，并与未标记的和FLAG标记的MYOC构建体进行比较。通过GLuc测定测量分泌的和可溶的细胞内MYOC eGLuc 2。在来自转染细胞的条件培养基中测定了9种另外的MYOC突变体的分泌，以测试该测定法用于监测其他MYOC变体的适用性。在分泌、可溶性细胞内水平和对药物治疗的反应方面，肌球蛋白eGLuc 2的表现与未标记和FLAG标记的MYOC相似。GLuc测定可在培养基更换后30分钟灵敏地检测Y 437 H MYOC分泌。Gaussia荧光素酶融合变体遵循预期趋势;非致病性变体（D208 E、G244 V）以野生型（WT）水平分泌，而预测的致病性变体（C245 Y、G246 R、E300 K、Y 437 H、I477 N）表现出实质性分泌缺陷。由C245 Y、G246 R和Y 437 H突变引起的分泌缺陷部分被允许的生长温度挽救。然而，有趣的是，这种分泌的增加与新合成的蛋白质无关。eGLuc 2与MYOC的融合不会显著改变MYOC的行为。这种新开发的MYOC报告系统可用于研究工程化的MYOC变体，并有可能鉴定MYOC分泌和功能的调节剂。
Primary open angle glaucoma–associated mutations in myocilin (MYOC) cause protein “nonsecretion,” rendering secreted MYOC difficult to detect using conventional techniques. This study focused on developing and using an assay that can quickly and easily detect mutant MYOC secretion. We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T and NTM-5 cells. Secreted and intracellular levels of MYOC eGLuc2 variants were evaluated by Western blotting and compared to untagged and FLAG-tagged MYOC constructs. Secreted and soluble intracellular MYOC eGLuc2 were measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from transfected cells to test the applicability of the assay for monitoring other MYOC variants. Myocilin eGLuc2 behaved similarly to untagged and FLAG-tagged MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 minutes after media change. Gaussia luciferase fused variants followed anticipated trends; nonpathogenic variants (D208E, G244V) were secreted at wild-type (WT) levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperature. Interestingly, however, this increase in secretion was independent of newly synthesized protein. Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. This newly developed MYOC reporter system can be used to study engineered MYOC variants and potentially to identify modulators of MYOC secretion and function.
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影响因子：
3.7
作者：
Badr CE;Hewett JW;Breakefield XO;Tannous BA
通讯作者：
Tannous BA
影响因子：
7.4
作者：
Maguire, Casey A.;Deliolanis, Nikolaos C.;Pike, Lisa;Niers, Johanna M.;Tjon-Kon-Fat, Lee-Ann;Sena-Esteves, Miguel;Tannous, Bakhos A.
通讯作者：
Tannous, Bakhos A.
影响因子：
--
作者：
Fan, BJ;Leung, DYL;Pang, CP
通讯作者：
Pang, CP
影响因子：
3.4
作者：
Annie Nguyen;Hulleman, John D.
通讯作者：
Hulleman, John D.
DOI：
10.1006/bbrc.2001.4624
发表时间：
2001-04-06
影响因子：
3.1
作者：
Caballero, M;Borrás, T
通讯作者：
Borrás, T