Department of Pediatrics, University of Chicago, Chicago, IL.
Mol Vis. 2020 Mar 18;26:204-215. eCollection 2020.
p62/Sequestosome 1 (p62) is a stress-induced protein that is involved in several different intracellular pathways, including regulation of aspects of protein degradation. p62 levels are elevated in several types of cataracts. We investigated whether levels of p62 and its phosphorylation were altered in the lenses of Cx50D47A mice, which express a mutant of connexin50 (Cx50) that leads to cataracts and impaired lens differentiation. To evaluate the importance of p62 in the lens defects caused by a connexin50 mutant, we also examined the effect of deleting in homozygous Cx50D47A mice.
Protein levels were determined with immunoblotting. Mouse lenses were examined with dark-field illumination microscopy. Intensities of the opacities and lens equatorial diameters were quantified using ImageJ. Nuclei and nuclear remnants were detected with fluorescence microscopy of lens sections stained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI).
Levels of total p62 were increased in the lenses of homozygous Cx50D47A mice compared to those of the wild-type animals. The ratio of p62 phosphorylated at threonine-269/serine-272 (T269/S272) to total p62 was significantly decreased, whereas the ratio of p62 phosphorylated at serine-349 (S349) to total p62 was significantly increased in lenses of homozygous Cx50D47A mice. However, deletion of did not affect the sizes of the lenses or the severity of their cataracts in homozygous Cx50D47A mice. Deletion of did not improve connexin50 or connexin46 levels. Moreover, deletion of did not change the levels of crystallins, histone H3, the mitochondrial import receptor subunit TOM20 homolog, or the abundance of nuclei and nuclear fragments in the lenses of homozygous Cx50D47A mice. Homozygous deletion of led to an 84% increase in the levels of ubiquilin 2, but did not significantly affect the levels of ubiquilin 1 or ubiquilin 4.
Although homozygous Cx50D47A lenses have increased levels of p62, a specific reduction in p62 phosphorylation at T269/S272, and a specific increase in p62 phosphorylation at S349, this protein is not a critical determinant of the severity of the abnormalities of these lenses (reduced growth or differentiation and cataracts). The lens may utilize redundant or compensatory systems (such as changes in levels of ubiquilin 2) to compensate for the lack of p62 in homozygous Cx50D47A lenses.
p62/自噬体相关蛋白 1（p62）是一种应激诱导蛋白，参与多种细胞内途径，包括调节蛋白降解的某些方面。p62 的水平在几种类型的白内障中升高。我们研究了突变型连接蛋白 50（Cx50）导致白内障和晶状体分化受损的 Cx50D47A 小鼠晶状体中 p62 及其磷酸化水平是否发生改变。为了评估 p62 在连接蛋白 50 突变体引起的晶状体缺陷中的重要性，我们还检查了在纯合 Cx50D47A 小鼠中缺失的效果。
用免疫印迹法测定蛋白水平。用暗场照明显微镜检查小鼠晶状体。用 ImageJ 定量评估混浊的强度和晶状体赤道直径。用 4',6-二脒基-2-苯基吲哚二盐酸盐（DAPI）染色的晶状体切片荧光显微镜检测核和核残片。
与野生型动物相比，纯合 Cx50D47A 小鼠晶状体中的总 p62 水平升高。p62 磷酸化的 threonine-269/serine-272（T269/S272）与总 p62 的比值显著降低，而 p62 磷酸化的 serine-349（S349）与总 p62 的比值显著升高。然而，在纯合 Cx50D47A 小鼠中，缺失并不会影响晶状体的大小或白内障的严重程度。缺失不会改变连接蛋白 50 或连接蛋白 46 的水平。此外，缺失不会改变晶状体中晶体蛋白、组蛋白 H3、线粒体输入受体亚基 TOM20 同源物或核和核碎片的丰度。纯合缺失导致 ubiquilin 2 的水平增加 84%，但不会显著影响 ubiquilin 1 或 ubiquilin 4 的水平。
尽管纯合 Cx50D47A 晶状体中 p62 水平升高，p62 磷酸化 at T269/S272 特异性降低，p62 磷酸化 at S349 特异性增加，但该蛋白不是这些晶状体异常严重程度（生长或分化减少和白内障）的关键决定因素。晶状体可能利用冗余或补偿系统（如 ubiquilin 2 水平的变化）来补偿纯合 Cx50D47A 晶状体中 p62 的缺乏。
