Yu Run, Cruz-Soto Martha, Li Calzi Sergio, Hui Hongxiang, Melmed Shlomo
UCLA School of Medicine, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Room D3066, Los Angeles, California 90048, USA.
J Endocrinol. 2006 Oct;191(1):45-53. doi: 10.1677/joe.1.06885.
Human pituitary tumor-transforming gene 1 (PTTG1) encodes a securin protein critically important in regulating chromosome separation. Murine PTTG (mPTTG) is 66% homologous to human PTTG1 and PTTG-null (PTTG-/-) mice exhibit pancreatic beta-cell hypoplasia and abnormal nuclear morphology with resultant diabetes. As we show that ductal beta-cell neogenesis is intact in PTTG-/- mice, we explored mechanism for defective beta-cell replication. We tested whether mPTTG exhibits securin properties in mouse insulin-secreting insulinoma MIN6 cells, using a live-cell system to monitor mitosis in cells transfected with an enhanced green fluorescent protein (EGFP)-tagged mPTTG conjugate (mPTTG-EGFP). To fulfill the criteria for securin properties, the protein should undergo degradation immediately before the metaphase-to-anaphase transition when expression levels are low, and should inhibit metaphase-to-anaphase transition when expression levels are high. EGFP itself did not undergo degradation throughout mitosis and high levels of EGFP per se did not affect normal mitosis progression (n=25). However, mPTTG-EGFP was degraded 2 min before the metaphase-to-anaphase transition when expression levels were low (n=19), and high mPTTG-EGFP levels blocked metaphase-to-anaphase transition in 13 cells. mPTTG-EGFP inhibited MIN6 cell proliferation and caused apoptosis. Immunocoprecipitation demonstrated binding of mPTTG-EGFP and separase. These results show that mPTTG exhibits properties consistent with a murine securin in insulin-secreting mouse cells and mPTTG overexpression inhibits cell proliferation, suggesting that defective beta-cell proliferation observed in PTTG-/- mice is likely due to abnormal cell-cycle progression.
人类垂体肿瘤转化基因1（PTTG1）编码一种在调节染色体分离中起关键作用的分离酶抑制蛋白。小鼠PTTG（mPTTG）与人类PTTG1有66%的同源性，PTTG基因敲除（PTTG-/-）小鼠表现出胰腺β细胞发育不全和异常的核形态，进而导致糖尿病。由于我们发现PTTG-/-小鼠的导管β细胞新生是完整的，因此我们探究了β细胞复制缺陷的机制。我们使用活细胞系统监测转染了增强型绿色荧光蛋白（EGFP）标记的mPTTG偶联物（mPTTG-EGFP）的小鼠胰岛素分泌瘤MIN6细胞中的有丝分裂，以测试mPTTG在小鼠胰岛素分泌细胞中是否具有分离酶抑制蛋白的特性。为了符合分离酶抑制蛋白特性的标准，该蛋白应在中期到后期转换前，当表达水平较低时立即降解，而当表达水平较高时应抑制中期到后期的转换。EGFP在整个有丝分裂过程中都不会降解，并且高水平的EGFP本身不会影响正常的有丝分裂进程（n = 25）。然而，当表达水平较低时，mPTTG-EGFP在中期到后期转换前2分钟被降解（n = 19），而高水平的mPTTG-EGFP在13个细胞中阻止了中期到后期的转换。mPTTG-EGFP抑制MIN6细胞增殖并导致细胞凋亡。免疫沉淀证明了mPTTG-EGFP与分离酶的结合。这些结果表明，mPTTG在胰岛素分泌小鼠细胞中表现出与小鼠分离酶抑制蛋白一致的特性，并且mPTTG过表达抑制细胞增殖，这表明在PTTG-/-小鼠中观察到的β细胞增殖缺陷可能是由于异常的细胞周期进程所致。
