Structure/Function/Relationship at Single Residue Resolution of the FcRn Transmembrane and Tail

项目总结 这项建议的目标是绘制快速回收的FCG的蛋白质-结构-功能关系图。 受体FcRN；并解释其生物学特性。 FcRN Tm和胞浆尾部高度保守的序列表明与FcRN的结构相互作用 FcRN功能所必需的膜。我们建议利用大规模寡核苷酸的进展进行研究 合成、多参数流式细胞仪和下一代测序以定量确定贡献 FcRN结构对免疫球蛋白循环的影响。我们将利用为Illumina启用的慢病毒FcRN表达文库 深度测序。该文库包括TM或胞质FcRN结构域中的非偏向突变，这些突变 将19个不同的氨基酸替换为每个天然残基(总共67个残基)-而不是 将剩余的蛋白质保留为野生型。我们将有数千个细胞表达每个突变 与WT相比，突变体回收免疫球蛋白的能力的比较将确定 突变。我们将使用这种新方法来测试假设驱动的突变，以描绘出 TM区域，有助于有效的内体循环和跨细胞作用。膜的局部化 蛋白质会受到TM长度等特性的影响。我们还将确定FcRN是否使用不同的 二聚化基序，在功能二聚体之间切换，就像对其他蛋白质所显示的那样。我们将使用这个 描述高度保守的细胞质结构域中的单个残基、基序或连接的技术 帮助有效的内体循环和跨细胞作用。研究表明，膜旁的两亲性螺旋 可能会感觉到/诱发弯曲。也有几个小的可能基序(YxxΦ，酸性二亮氨酸，CaM结合， 磷酸化位点)，这将通过系统的螺旋插入/缺失/扰乱来探测。我们将表演 这种慢病毒跨细胞类型的筛查；这些结果的比较将提供有关一般性的信息 或突变命中的专一性。无论是假说驱动的突变，还是来自非 有偏见的突变将通过我们实验室使用的循环和跨细胞分析来进一步表征。快感-- 制作的分辨率地图将为外业提供广泛的实用程序，作为询问 任何蛋白质中的膜-结构/功能关系，其中功能服从于基于流式细胞仪的 单元格读出和排序。同样重要的是，这个项目将扩大我在细胞和膜生物学方面的培训 与人类疾病相关，并使我能够有意义地扩展我在生命科学领域的职业生涯。

PROJECT SUMMARY The goal of this proposal is to map the protein-structure-functional relationships of the rapidly recycling Fcg- receptor FcRn; and explain its biology. Highly conserved sequences in the TM and cytosolic tail of FcRn indicate structural interactions with the membrane necessary for FcRn function. We propose studies utilizing advances in large-scale oligonucleotide synthesis, multi-parameter FACS, and next-generation sequencing to quantitatively determine the contribution of FcRn structure to IgG recycling. We will utilize a lentiviral FcRn-expression library that is enabled for Illumina deep sequencing. The library includes non-biased mutations in the TM or cytosolic FcRn domains that incorporate 19 different amino acid substitutions for every native residue (67 residues in total) - while otherwise keeping the rest of the protein as wild type. We will have several thousand cells expressing each mutation and comparison of the mutants ability to recycle IgG, as compared to WT, will determine the significance of the mutation. We will use this novel method to test hypothesis-driven mutations to delineate structural features in the TM region that contribute to efficient endosome recycling and transcytosis. Localization of membrane proteins can be influenced by properties such as TM length. We will also determine if FcRn use different dimerization motifs, to switch between functional dimers, as has been shown for other proteins. We will use this technology to delineate individual residues, motifs, or linkages in the highly conserved cytoplasmic domain that assists efficient endosome recycling and transcytosis. Research suggests an juxtamembrane amphipathic helix may sense/induce curvature. There are also several small likely motifs (YXXΦ, acidic di-leucine, CaM-binding, phosphorylation sites) that will be probed by systematic helix insertions/deletions/scrambling. We will perform this lentiviral screen across cell types; the comparison of those results will provide information on the generality or specificity of the mutational hits. Both the hypothesis-driven mutations, and the discoveries from the non- biased mutations will be further characterized with recycling and transcytosis assays used in our lab. The high- resolution map produced will provide broad utility for the field, serving as a template by which to interrogate the membrane-structure/function relationships that govern in any protein where function is amenable to FACS-based single-cell readout and sorting. As importantly, this project will expand my training in cell and membrane biology relevant to human diseases and put me in a position to meaningfully extend my career in the life sciences.