Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency
Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency
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DOI：
10.1073/pnas.0914019107
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发表时间：
2010-02-23
影响因子：
11.1
通讯作者：
Lesley, Scott A.
中科院分区：
文献类型：
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作者：
Gonzalez, Rodolfo;Jennings, Lori L.;Lesley, Scott A.
据预测，大约3，500个哺乳动物基因是分泌的或单次跨膜蛋白。这些基因中的大多数的功能仍然是未知的，并且许多编码的蛋白质本身可能被用作新的治疗剂或作为小分子或抗体药物开发的靶点。为了分析胞外蛋白质组的生理活性，我们开发了一个大规模、高通量的蛋白质表达、纯化和筛选平台。在这项研究中，完整的人类细胞外蛋白质组进行了分析，并根据全基因组疾病关联研究的优先顺序，以选择529个初始靶基因。将这些基因作为天然序列以及作为N-末端和C-末端Fc融合体克隆到三个表达载体中，以产生806个纯化分泌蛋白的初始集合。为了确定其效用，在基于OCT 4的细胞测定中筛选该文库以鉴定人胚胎干细胞自我更新的调节剂。我们发现色素上皮衍生因子可以促进人胚胎干细胞的长期多能性生长，而无需bFGF或TGF β/Activin/Nodal配体补充。我们的研究结果进一步表明，激活色素上皮衍生因子受体-Erk 1/2信号通路的色素上皮衍生因子是足以维持自我更新的多能人类胚胎干细胞。这些实验说明了通过在基于细胞的表型或报告基因测定中直接筛选蛋白质多样性来发现新的生物学功能的潜力。
Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGF beta/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.