Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein.
Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein.
复制标题
DOI：
10.3390/ijms140715121
复制
发表时间：
2013-07-22
影响因子：
5.6
通讯作者：
Bhattacharya SS
中科院分区：
文献类型：
--
作者：
Doumanov JA;Zeitz C;Dominguez Gimenez P;Audo I;Krishna A;Alfano G;Diaz ML;Moskova-Doumanova V;Lancelot ME;Sahel JA;Nandrot EF;Bhattacharya SS
BEST 1基因编码的雌激素样肽-1（Best 1）蛋白的突变与黄斑营养不良有关。Best 1主要在视网膜色素上皮（RPE）中表达，并插入其基底外侧膜中。我们研究了疾病相关Best 1突变蛋白在极化MDCKII细胞中的细胞定位，以研究Best 1的特异性分选基序。进行MDCK细胞中BEST 1内源性表达的实时PCR和蛋白质印迹。使用定点诱变产生Best 1突变体构建体并转染MDCK细胞。对于蛋白质分选，使用共聚焦显微镜研究、生物素化测定和用于定量错误定位的统计方法。BEST 1在MDCK细胞中的内源性表达的分析揭示BEST 1转录物的存在，但没有蛋白质。共聚焦显微镜和定量分析表明，转染的正常人Best 1显示在MDCK细胞的基底外侧定位，而几个Best 1突变体（Y85 H，Q96 R，L100 R，Y227 N，Y227 E）的细胞分选被改变。与组成型活性的Y227 E相反，组成型非活性的Y227 F Best 1突变体定位于与正常Best 1蛋白相似的基底外侧。我们的数据表明，至少有三个基底外侧排序图案可能涉及适当的Best 1基底外侧定位。此外，非磷酸化的酪氨酸227可以发挥作用的基底外侧交付。
Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery.
登录
查看更多内容
影响因子：
3.8
作者：
Marmorstein, Lihua Y;Wu, Jiang;McLaughlin, Precious;Yocom, John;Karl, Mike O;Neussert, Rudgar;Wimmers, Soenke;Stanton, J Brett;Gregg, Ronald G;Strauss, Olaf;Peachey, Neal S;Marmorstein, Alan D
通讯作者：
Marmorstein, Alan D
影响因子：
17.8
作者：
Marmorstein, Alan D.;Cross, Harold E.;Peachey, Neal S.
通讯作者：
Peachey, Neal S.
影响因子：
9.2
作者：
Matter, K
通讯作者：
Matter, K
影响因子：
4.4
作者：
Guziewicz, Karina E.;Zangerl, Barbara;Aguirre, Gustavo D.
通讯作者：
Aguirre, Gustavo D.
影响因子：
4.4
作者：
Bakall, B;Marmorstein, LY;Marmorstein, AD
通讯作者：
Marmorstein, AD