Couderc Bettina, Penary Marie, Tohfe Mustapha, Pradines Anne, Casteignau Antoine, Berg Danièle, Favre Gilles
INSERM U563, Department Innovations thérapeutiques et Oncologie moléculaire, Institut Claudius Regaud and Faculté des Sciences Pharmaceutiques, Toulouse, France.
BMC Biotechnol. 2006 May 29;6:26. doi: 10.1186/1472-6750-6-26.
The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function.
We constructed an adenoviral vector coding a chimeric p53 protein by fusing p53 sequence with the 21 COOH term amino acids sequence of H-Ras. Indeed, the translation products of Ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a CAAX motif present at the C terminus of Ras proteins. The chimeric p53HRCaax protein was farnesylated efficiently in transduced human osteosarcoma p53-/- cell line. The farnesylated form of p53 resided mainly in the cytosol, where it is non-functional. Farnesyl transferase inhibitors (FTIs) specifically inhibited farnesyl isoprenoid lipid modification of proteins. Following treatment of the cells with an FTI, p53HRCaax underwent translocation into the nucleus where it retained transcription factor activity. Shifting p53 into the nucleus resulted in the induction of p21waf1/CIP1 and Bax transcription, cell growth arrest, caspase activation and apoptosis.
Artificial protein farnesylation impaired the transcriptional activity of p53. This could be prevented by Farnesyl transferase inhibition. These data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor.
在基因治疗临床试验中使用整合型病毒载体已指出异位基因整合到细胞基因组的有害影响问题以及该策略的安全性问题。我们在此提出一种方法，通过作用于促凋亡蛋白的细胞定位及其凋亡功能的激活，在需要时诱导基因修饰细胞死亡。
我们构建了一种腺病毒载体，通过将p53序列与H-Ras的21个COOH末端氨基酸序列融合来编码嵌合p53蛋白。实际上，Ras基因的翻译产物是胞质蛋白，通过由Ras蛋白C末端存在的CAAX基序引发的一系列翻译后修饰，这些蛋白会与膜发生二次结合。嵌合p53HRCaax蛋白在转导的人骨肉瘤p53 - / - 细胞系中被有效法尼基化。法尼基化形式的p53主要驻留在胞质溶胶中，在那里它没有功能。法尼基转移酶抑制剂（FTIs）特异性抑制蛋白质的法尼基类异戊二烯脂质修饰。用FTI处理细胞后，p53HRCaax易位到细胞核中，在那里它保留转录因子活性。将p53转移到细胞核中导致p21waf1/CIP1和Bax转录的诱导、细胞生长停滞、半胱天冬酶激活和细胞凋亡。
人工蛋白质法尼基化损害了p53的转录活性。这可以通过抑制法尼基转移酶来预防。这些数据突出了这样一个事实，即蛋白质的人工异戊二烯化提供了一种控制反式激活因子功能的新系统。
