Departments Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY, 10461, USA; Genetics, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.
Department of Biochemistry & Molecular Biology, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR, 97239, USA.
Exp Eye Res. 2019 Feb;179:32-46. doi: 10.1016/j.exer.2018.10.011. Epub 2018 Oct 22.
Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular compositions of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein compositions between the lens epithelium and fibers, we employed tandem mass spectrometry (2D-LC/MS) analysis of microdissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular processes and subcellular localizations, were adapted for the lens. Expression levels of both epithelial and fiber proteomes were compared with whole lens proteome and mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins (e.g. Carhsp1), translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal [e.g. non-muscle myosin IIA heavy chain (Myh9) and βB2-spectrin (Sptbn2)] and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown functions in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets represent the first mouse lens epithelium and fiber cell proteomes, establish comparative analyses of protein and RNA-Seq data, and characterize the major proteome remodeling required to form the mature lens fiber cells.
上皮细胞和分化的纤维细胞代表了眼晶状体中的不同隔室。虽然之前的研究已经揭示了在上皮细胞与纤维细胞中优先表达的蛋白质，但比较上皮细胞与纤维细胞的分子组成的综合蛋白质组学文库对于理解晶状体的形成、功能、疾病和再生潜能以及有效分化多能干细胞用于体外晶状体发育和病理学建模是至关重要的。为了比较晶状体上皮细胞和纤维细胞之间的蛋白质组成，我们采用了串联质谱（2D-LC/MS）分析从小鼠 P0.5 晶状体中微分离的细胞。通过分子过程和亚细胞定位，将前 525 种鉴定出的蛋白质的功能分类归入基因本体论类别，适用于晶状体。使用 E14.5、E16.5、E18.5 和 P0.5 RNA-Seq 数据集比较上皮细胞和纤维细胞蛋白质组的表达水平与整个晶状体蛋白质组和 mRNA 水平。在这个发育时间窗口内，多个复杂的生物合成和分解代谢过程为晶状体的透明度生成了分子和结构基础。正如预期的那样，晶体蛋白的 mRNA 和蛋白质水平之间存在高度相关性。综合数据分析证实和/或预测了转录因子（TFs）、RNA 结合蛋白（例如 Carhsp1）、翻译装置（包括核糖体异质性和起始因子）、微管、细胞骨架（例如非肌肉肌球蛋白 IIA 重链（Myh9）和βB2- spectrin（Sptbn2））和膜蛋白在晶状体形成和成熟中的作用。我们的数据突出了许多在晶状体中具有未知功能的蛋白质，这些蛋白质在上皮细胞或纤维细胞中优先富集，为进一步研究这些蛋白质在纤维细胞分化与上皮细胞维持中的作用奠定了基础。总之，本蛋白质组数据集代表了第一个小鼠晶状体上皮细胞和纤维细胞蛋白质组，建立了蛋白质和 RNA-Seq 数据的比较分析，并描述了形成成熟晶状体纤维细胞所需的主要蛋白质组重排。
