Inhibition of prion amplification by expression of dominant inhibitory mutants--a systematic insertion mutagenesis study.
Inhibition of prion amplification by expression of dominant inhibitory mutants--a systematic insertion mutagenesis study.
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通过显性抑制突变体的表达抑制朊病毒扩增——系统插入诱变研究。
DOI：
10.2174/1871526510909010040
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
M. Groschup
中科院分区：
文献类型：
--
作者：
M. Geissen;Harriet Mella;A. Saalmüller;M. Eiden;J. Proft;E. Pfaff;H. Schätzl;M. Groschup
到目前为止，还不清楚Prion蛋白(PrP)的哪些结构元件参与其转换过程。这些关键区域的特征将有助于理解转换过程本身，并可能有助于开发特定的治疗方法，通过显性抑制突变来抑制PrP(RES)的形成。为了解决这个重要的问题，我们产生了33个均匀分布的插入突变体，覆盖了小鼠3F4标记的PrP的整个序列。用逆转录病毒转导的方法在3种不同的瘙痒病感染细胞系(SCN2A、SMB[RC040]、SMB[22F])中表达突变体。可转换性不仅通过在假定的折叠区域(aa100-170)中引入插入而受到影响，而且在PrP的C末端(最高可达aa214)中也受到影响。此外，在四个显著区域(aa100-112、aa130-154、aa166-172、aa196-200)观察到PrP突变体对转化的显性抑制作用。基于计算机的结构分析表明，这些片段被组织在两个结构上明显分开的区域，支持它们可以作为种子形成过程中必需的蛋白质-蛋白质相互作用位点的想法。
Until now it is still not clear which structural elements of the prion protein (PrP) are involved in its conversion process. Characterisation of these essential regions would help to understand the conversion process itself and might help to develop specific therapeutic approaches to inhibit PrP(res) formation by dominant inhibitory mutations. To address this important question 33 evenly spaced insertion mutants were generated spanning the entire sequence of the murine 3F4-tagged PrP. The mutants were expressed by retroviral transduction in three different scrapie infected cell lines (ScN2a; SMB[RC040]; SMB[22F]). The convertibility was affected not only by introducing the insertion in the putatively refolded region (aa100-170), but also in the C-terminus of PrP (up to aa214). Moreover, dominant inhibitory effects on conversion were observed for PrP-mutants at four distinguished regions (aa100-112; aa130-154; aa166-172, aa196-200). Computer based structural analysis revealed that these segments were organized in two structurally clearly separated regions supporting the idea that they could function as protein-protein interaction sites which are necessary during seed formation.