Mammalian L1 retrotransposon replication

最新发现： ORF 1 p的结构与功能研究-ORF 1 p是L1基因编码的两种蛋白质之一。其他人对小鼠ORF 1 p的早期研究表明，它结合核酸，作为核酸伴侣，并通过高度保守的卷曲螺旋结构域形成三聚体。然而，ORF 1 p在逆转录转座中的功能在很大程度上是未知的。我们正在使用几种方法来研究这个问题，包括分析正选择的结构、生物化学和生物学效应，其主要涉及卷曲螺旋结构域（Boissinot等人，Mol.生物学评价18：2186）。为此，我们从已灭绝的L1家族L1 Pa 5中复活了一个ORF 1 p，它是目前活跃的人类L1 Pa 1家族的现代人（h）ORF 1 p的祖先。我们还创建了包含现代和祖先区域的ORF 1 p的马赛克版本，以及删除各种结构域的其他变体。我们广泛的特点，这些蛋白质的反转录转座和相互作用与几种哺乳动物宿主蛋白在体内。此外，我们纯化了mg量的各种ORF 1 ps至均一。我们在体外研究了它们的各种生物物理和生物化学特性，包括反映它们的核酸伴侣活性的几种测定。我们在2012年发现了ORF 1 p的两个新特性：hORF 1 p三聚体可以在高亲和力核酸结合所需的条件下可逆地聚合，并且这种特性涉及蛋白质的第二个新特性，即其错配双链核酸的双相效应，保护其在低浓度下免于解离（熔化），但在高浓度下熔化它主要是聚合物。错配的双链体是代理核酸伴侣底物。因此，确定生物物理基础上的蛋白质对该底物的令人惊讶的和新的双相影响是必不可少的，我们在这一领域的进一步进展。因此，在2013年初，我们开始与Mark威廉姆斯博士合作，通过原子力显微镜研究这些相互作用。今年，还实施了一种新的灵敏的FRET测定法，用于评估平衡条件下的核酸伴侣活性，对卷曲螺旋结构域进行了广泛的突变分析，试图阐明蛋白质这一区域中正选择的生化作用，并确定ORF 1 p在我们进行突变分析的许多位点被磷酸化。在过去的一年里，我们在这两个项目上都取得了重大进展：关于卷曲螺旋，我们发现，将现代适应性卷曲螺旋的4个氨基酸中的任何一个改变为它们的祖先对应物消除了反转录转座，并且只有通过将卷曲螺旋中的17个额外的现代残基转换为它们的祖先对应物来恢复活性，才能恢复活性。需要协同变化来补偿单个氨基酸变化的影响，这为卷曲螺旋进化过程中的正选择提供了理论基础，并表明卷曲螺旋不同区域之间的串扰可以延伸很长的距离，对于维持功能结构至关重要。确定这种串扰的基础可以提供关于三聚体结构如何与其在逆转录转座中的作用相关的重要见解。关于磷酸化的研究，我们刚刚向PNAS提交了一篇论文，报告说L1复制依赖于ORF 1 p的磷酸化。因此，L1与宿主调控系统整合，并可能竞争和干扰宿主调控系统，这些系统负责许多重要的宿主过程，如细胞分裂和分化。这些发现不仅代表了我们对L1调节的理解的重大进展，而且还表明L1对其宿主的影响可能远远超过遗传变化的作用。

RECENT FINDINGS: STRUCTURAL AND FUNCTIONAL STUDIES OF ORF1p - ORF1p is one of two L1 encoded proteins. Earlier studies by others of mouse ORF1p showed that it binds nucleic acids, acts as a nucleic acid chaperone, and forms trimers via a highly conserved coiled coil domain. However, the function of ORF1p in retrotransposition is largely unknown. We are using several approaches to examine this problem including analysis of the structural, biochemical, and biological effects of positive selection, which involved mainly the coiled coil domain (Boissinot, et al, Mol. Biol. Evol. 18: 2186). To do so we resuscitated an ORF1p from an extinct L1 family, L1Pa5, which is ancestral to the modern human(h) ORF1p of the currently active human L1Pa1 family. We also created mosaic versions of ORF1p that contain modern and ancestral regions, and other variants that were deleted of various domains. We extensively characterized these proteins with respect to retrotransposition and interaction with several mammalian host protein in vivo. In addition, we purified mg amounts of some the various of ORF1ps to homogeneity. We examined their various biophysical and biochemical properties in vitro including several assays that reflect their nucleic acid chaperone activity. We uncovered two novel properties of ORF1p in 2012: That hORF1p trimers can reversibly polymerize under the conditions required for high affinity nucleic acid binding, and that this property is involved in the second novel property of the protein, namely its biphasic effect of mismatched double-stranded nucleic acids, protecting it from dissociation (melting) at low concentrations, but melting it at high concentrations largely polymeric. A mismatched duplex is a proxy nucleic acid chaperone substrate. Thus, determining the biophysical basis the surprising and novel diphasic affect of the protein on this substrate is essential to our further progress in this area. Thus, in early 2013 we began a collaboration with Dr. Mark Williams to study these interactions by atomic force microscopy. This year, also implemented a new sensitive FRET assay for assessing nucleic acid chaperone activity under equilibrium conditions, carried out extensive mutational analysis of coiled coil domain in an attempt to clarify the biochemical effect of positive selection in this region of the protein, and determined that ORF1p is phosphorylated at a number of sites which we subjected to mutational analysis. Over the past year we made significant progress on both projects: With regard to the coiled coil we found that change of any one of just 4 amino acids of a modern adapted coiled coil to their ancestral counterparts eliminate retrotransposition and that activity is only restored by converting 17 additional modern residues in the coiled coil to their ancestral counterparts to restore activity. That concerted changes are required to compensate for the effects of single amino acid changes provides a rationale for positive selection during evolution of the coiled coil and indicates that cross talk between different regions of the coiled coil can extend over long distance and is crucial for maintaing a functional structure. Determining the basis of this cross talk could offer major insights as to how the trimeric structure is related to its role in retrotransposition. With regard to the phosphorylation studies we just submitted a paper to PNAS which reports that L1 replication depends on phosphorylation ofORF1p. Thus L1 is integrated with, and perhaps competes for and perturbs the host regulatory systems responsible for numerous essential host processes such as cell division and differentiation. These findings represent not only a major advance in our understanding of L1 regulation but also suggest that the effect of L1 on its host is likely far more profound than just an agent of genetic change.