Functional specific roles of H-ras and N-ras. A proteomic approach using knockout cell lines
Functional specific roles of H-ras and N-ras. A proteomic approach using knockout cell lines
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DOI：
10.1002/elps.201100606
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发表时间：
2012-05-01
期刊：
影响因子：
2.9
通讯作者：
Martinez-Salgado, Carlos
中科院分区：
文献类型：
--
作者：
Ferreira, Laura;Fuentes-Calvo, Isabel;Martinez-Salgado, Carlos
Ras小GTP酶作为细胞外信号的转导子调节细胞存活、生长和分化。有三种主要的ras亚型：H-、N-和K-Ras。为了提高对H-和N-Ras蛋白信号传导网络的理解，我们比较了H-ras和/或N-ras基因敲除小鼠胚胎成纤维细胞中总蛋白质组的变化，使用蛋白质组学工具结合2DE，半定量图像分析，凝胶内胰蛋白酶消化和质谱。由于H-Ras的表达缺失，有四种蛋白表达上调（包括细胞周期蛋白依赖性激酶抑制剂2A）和8个下调的（包括应激-70蛋白、二氢嘧啶酶相关蛋白3、热休克同源71 kDa蛋白、原肌球蛋白β链、Rho GDP解离抑制剂1）和六种上调蛋白（例如白细胞弹性蛋白酶抑制剂A、L-乳酸脱氢酶B链、c-Myc应答蛋白Rcl、白细胞介素-1受体拮抗剂蛋白）。这些蛋白质中的大多数以这样或那样的方式与Ras信号传导相关。通过Western blot进一步证实了这些蛋白中的一些蛋白的表达变化。从H-和N-Ras敲除成纤维细胞功能丧失的蛋白质组学比较分析产生可解释的数据，以阐明差异蛋白质表达，并有助于评估生理和治疗靶点的可能性。
Ras small GTPases function as transducers of extracellular signals regulating cell survival, growth and differentiation. There are three major ras isoforms: H-, N- and K-Ras. To improve the understanding of H- and N-Ras protein signalling networks, we compared total proteome changes in mouse embryonic fibroblasts knock out for H-ras and/or N-ras, using proteomics tools combining 2DE, semi-quantitative image analysis, in-gel trypsin digestion and mass spectrometry. There are four up-regulated proteins due to the loss of expression of H-Ras (including cyclin-dependent kinase inhibitor 2A) and eight down-regulated (including stress-70 protein, dihydropyrimidinase-related-protein 3, heat shock cognate 71 kDa protein, tropomyosin beta chain, Rho GDP-dissociation inhibitor 1) and six up-regulated proteins (e.g. leukocyte elastase inhibitor A, L-lactate dehydrogenase B chain, c-Myc-responsive protein Rcl, interleukin-1 receptor antagonist protein) due to the loss of expression of both N- and H-Ras. Most of these proteins are related to Ras signalling in one way or another. Changes in expression of some of these proteins were further confirmed by Western blot. This proteomic comparative analysis from loss of function of H- and N-Ras knockout fibroblasts yields interpretable data to elucidate the differential protein expression, and contributes to evaluate the possibilities for physiological and therapeutic targets.