背景:R-RAS2是一种在调节和信号水平上与经典RAS蛋白高度相似的小GTP酶。R-RAS2的高度进化保守性,它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R-RAS2的结构和功能特性。
方法:生物信息学分析用于阐明R-RAS蛋白的进化。用GTP酶-GloTM测定试剂盒分析纯化的人和海绵蛋白的固有GTP酶活性。细胞模型由用EsuRRAS2样或HsaRRAS2瞬时转染的人乳腺癌细胞系MCF-7和MDA-MB-231组成。通过蛋白质印迹对全细胞裂解物或细胞粘附蛋白分离物进行R-RAS2蛋白的生物学表征,免疫荧光和共聚焦显微镜,MTT试验,集落形成试验,伤口愈合和Boyden室迁移试验。
结果:我们发现单个海绵R-RAS2样基因/蛋白可能反映了在向Bilateria过渡期间重复之前存在的祖先R-RAS蛋白的特性,还有Vertebrata.海绵和人R-RAS2的生化表征表明它们具有相同的固有GTP酶活性和RNA结合特性。通过测试细胞增殖,MDA-MB-231人乳腺癌细胞的迁移和集落形成效率,我们发现R-RAS蛋白的祖先类型,类似海绵R-RAS2,增强了它们的致癌潜力,类似于人类R-RAS2。此外,海绵和人R-RAS2未在粘着灶中发现,但是两种同源物通过增加talin1和vinculin在其调节中起作用。
结论:这项研究表明,所有动物的祖先都拥有一种R-RAS2样蛋白,其致癌特性与进化上更新版本的蛋白相似,甚至在真实组织出现和肿瘤起源之前。因此,我们揭示了后生动物中R-RAS2的进化史,提高了我们对R-RAS2特性的认识,包括它的结构,调节和功能。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.