Newborn Mouse Lens Proteome and Its Alteration by Lysine 6 Mutant Ubiquitin

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. To study the developmental processes that require intact ubiquitin, we executed the most extensive characterization of the lens proteome to date. We quantified lens protein expression changes in multiple replicate pools of P1 wild-type and K6W-Ub-expressing mouse lenses. Lens proteins were digested with trypsin, peptides were separated using strong cation exchange and reversed-phase liquid chromatography, and tandem mass (MS/MS) spectra were collected with a linear ion trap. Transgenic mice that expressed low levels of K6W-Ub (low expressers) had normal, clear lenses at birth, whereas the lenses that expressed high levels of K6W-Ub (higher expressers) had abnormal lenses and cataracts at birth. A total of 2052 proteins were identified, of which 996 were reliably quantified and compared between wild-type and K6W-Ub transgenic mice. Consistent with a delayed developmental program, fiber-cell-specific proteins, such as γ-crystallins (γA, γB, γC, and γE), were down-regulated in K6W-Ub higher expressers. Up-regulated proteins were involved in energy metabolism, signal transduction, and proteolysis. The K6W-Ub low expressers exhibited delayed onset and milder cataract consistent with smaller changes in protein expression. Because lens protein expression changes occurred prior to lens morphological abnormalities and cataract formation in K6W-Ub low expressers, it appears that expression of K6W-Ub sets in motion a process of altered protein expression that results in developmental defects and cataract.泛素（ubiquitin）是一种分子标签，常于泛素蛋白酶体系统（ubiquitin proteasome system）中介导蛋白酶体对蛋白质的降解。在晶状体中靶向表达K6W突变型泛素（K6W-Ub）会引发晶状体发育缺陷与白内障形成，这提示泛素在晶状体发育中发挥关键功能。为探究完整泛素所参与的发育过程，我们完成了迄今最为全面的晶状体蛋白质组学表征工作。我们对多组重复的P1代野生型与K6W-Ub表达型小鼠晶状体样本的蛋白质表达变化进行了定量分析：首先将晶状体蛋白经胰蛋白酶消化，随后通过强阳离子交换色谱与反相液相色谱分离肽段，并采用线性离子阱采集串联质谱（MS/MS）谱图。表达低水平K6W-Ub的转基因小鼠（低表达组）出生时晶状体正常且透明；而表达高水平K6W-Ub的转基因小鼠（高表达组）出生时即出现晶状体异常与白内障。本研究共鉴定出2052种蛋白质，其中996种可被可靠定量，并在野生型与K6W-Ub转基因小鼠间进行了表达差异比较。与发育程序延迟的表型一致，纤维细胞特异性蛋白（如γ-晶状体蛋白γA、γB、γC与γE）在K6W-Ub高表达组中表达下调。表达上调的蛋白质则参与能量代谢、信号转导与蛋白水解过程。K6W-Ub低表达组的白内障发病延迟且症状更轻微，这与蛋白质表达变化幅度更小的结果相符。由于在K6W-Ub低表达组中，晶状体蛋白质表达变化先于晶状体形态异常与白内障形成，可见K6W-Ub的表达会启动蛋白质表达异常的进程，最终导致发育缺陷与白内障。