The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

中文翻译：

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R-RAS 蛋白的祖先类型具有致癌潜力

R-RAS2 是一种小型 GTP 酶，在调节和信号传导水平上与经典 RAS 蛋白高度相似。R-RAS2 的高度进化保守性、其与基本细胞过程的联系及其在癌症中的作用，使 R-RAS2 成为一个有趣的研究课题。为了阐明 R-RAS 蛋白的进化史，我们研究并比较了祖先型 R-RAS 蛋白与人类 R-RAS2 的结构和功能特性。生物信息学分析用于阐明 R-RAS 蛋白的进化。使用 GTPase-GloTM 检测试剂盒分析纯化的人和海绵蛋白的内在 GTP 酶活性。该细胞模型由瞬时转染 EsuRRAS2 样或 HsaRRAS2 的人乳腺癌细胞系 MCF-7 和 MDA-MB-231 组成。通过全细胞裂解物或细胞粘附蛋白分离物的蛋白质印迹、免疫荧光和共聚焦显微镜、MTT 测试、集落形成测定、伤口愈合和博伊登室迁移测定来进行 R-RAS2 蛋白的生物学表征。我们发现，单个海绵 R-RAS2 样基因/蛋白可能反映了祖先 R-RAS 蛋白的特性，该蛋白在向两侧对称动物和脊椎动物过渡期间复制之前就存在。海绵和人 R-RAS2 的生化特征表明，它们具有相同的内在 GTP 酶活性和 RNA 结合特性。通过测试 MDA-MB-231 人乳腺癌细胞的细胞增殖、迁移和集落形成效率，我们发现 R-RAS 蛋白的祖先类型（海绵 R-RAS2 样）增强了其致癌潜力，类似于人类 R -RAS2。此外，在粘着斑中未发现海绵和人R-RAS2，但这两种同源物通过增加talin1和vinculin在其调节中发挥作用。这项研究表明，即使在真正的组织出现和肿瘤起源之前，所有动物的祖先都拥有一种类似 R-RAS2 的蛋白质，其致癌特性与该蛋白质在进化上的更新版本相似。因此，我们揭开了R-RAS2在后生动物中的进化史，并提高了我们对R-RAS2特性（包括其结构、调控和功能）的认识。