Mammalian L1 retrotransposon replication

最新调查结果： ORF1p的结构和功能研究-ORF1p是逆转录转座所必需的两个L1编码蛋白之一。我们早些时候已经证明灵长类ORF1p(与小鼠相似)是一个螺旋螺旋介导的三聚体，具有核酸结合和伴侣活性。然而，ORF1p在逆转录转座中的作用在很大程度上还不清楚。在过去的一年里，我们在ORF1p的研究中取得了两个重要进展： (1)我们发现L1的活性需要高度保守的脯氨酸依赖的磷酸激酶(PDPK)位点的磷酸化。我们还发现，在HeLa细胞中表达的ORF1p可以与Pin1结合，Pin1是一种在磷酸化介导的事件中通过催化磷酸化PDPK位点的Pro顺反异构化而发挥关键作用的蛋白。可逆的蛋白质磷酸化是由蛋白激酶、磷酸酶和辅助蛋白相互作用的级联反应所影响的，是许多基本生物过程的调节和协调的支柱。因此，证明L1活性的磷酸化依赖性大大扩大了我们对L1/宿主相互作用范围的了解。 (2)与东北大学威廉姆斯实验室合作，我们利用单分子研究表明，带有突变螺旋线圈的ORF1p在逆转录转座中不活跃，但与具有非突变螺旋螺旋的活跃ORF1p相比，在活性蛋白质的比率为1/10时，在单链DNA上形成稳定结合的寡聚体。因此，齐聚速率是一个新的和关键的螺旋线圈依赖的ORF1p活性参数。我们还表明，这一特性一直是保守的，尽管在灵长类进化的最后25MyR期间，卷曲线圈发生了广泛的重塑，从而使代表卷曲线圈进化的重复的正选择事件合理化。

RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins that are essential for retrotransposition. We earlier showed that primate ORF1p (like that in mouse) is a coiled coil mediated trimer that has nucleic acid binding and chaperone activity. However, the function of ORF1p in retrotransposition is largely unknown. This past year we made two important advances in our studies of ORF1p: (1) We showed that L1 activity requires phosphorylation of highly conserved proline dependent phosphokinase (PDPK) sites. We also showed that ORF1p expressed in HeLa cells can bind Pin1, a protein that serves a critical function in phosphorylation mediated events by catalyzing cis / trans isomerization of proline at phosphorylated PDPK sites. Reversible protein phosphorylation is effected by interacting cascades of protein kinases, phosphatases, and ancillary proteins, and is a mainstay in the regulation and coordination of many basic biological processes. Therefore, demonstrating phosphorylation-dependence of L1 activity substantially enlarged our knowledge of the scope of L1 / host interaction. (2) In collaboration with the Williams laboratory at Northeastern University, we used single molecule studies to show that an ORF1p with a mutated coiled coil that is inactive in retrotransposition, but exhibits no significant biochemical differences from an active ORF1p with a non-mutated coiled coil, forms stably bound oligomers on ssDNA at <1/10th the rate of active proteins. Thus, oligomerization rate is a novel and critical coiled coil-dependent parameter of ORF1p activity. We also showed that this property has been conserved, despite extensive remodeling of the coiled coil during the last 25 Myr of primate evolution, thus rationalizing the repeated episodes of positive selection that typifies coiled coil evolution.