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

FcRn 跨膜和尾部单残基分辨率的结构/功能/关系

• 批准号: 10152591
• 负责人: Jamie S LeBarron
• 金额: $ 0.61万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2021-05-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152591
• 关键词: Affect; Albumins; Amino Acid Substitution; Behavior; Binding; Biological Assay; Biological Sciences; Biology; Cell Differentiation process; Cell Line; Cell Lineage; Cells; Cellular biology; Conserved Sequence; Coupled; Custom; Cytoplasmic Tail; Dimerization; Endosomes; Endothelial Cells; Endothelium; Epithelial; Epithelial Cells; Eukaryotic Cell; Expression Library; Goals; Half-Life; Hela Cells; Host Defense; Human; Immunoglobulin G; Immunologic Surveillance; Individual; Integral Membrane Protein; Laboratories; Length; Leucine; Libraries; Lipid Bilayers; Lipids; Lysosomes; Mammals; Maps; Membrane; Membrane Biology; Membrane Proteins; Membrane Structure and Function; Methods; Modernization; Molecular Conformation; Movement; Mucous Membrane; Mutation; Oligonucleotides; Pathway interactions; Peptides; Phosphorylation Site; Positioning Attribute; Postdoctoral Fellow; Proline; Property; Proteins; Recycling; Research; Resolution; Rest; Schools; Site; Sorting - Cell Movement; Specificity; Structure; Structure-Activity Relationship; Surface; Tail; Technology; Testing; Thick; Training; Transmembrane Domain; Work; base; career; cell type; deep sequencing; design; dimer; exhaustion; experimental study; human disease; insertion/deletion mutation; insight; interest; late endosome; macrophage; membrane model; mutant; neonatal Fc receptor; next generation sequencing; novel; protein function; protein structure; protein structure function; receptor; receptor recycling; trafficking; transcytosis; uptake

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.

项目摘要 该提案的目标是绘制快速循环Fcg的蛋白质-结构-功能关系， 受体FcRn;并解释其生物学。 在TM和FcRn的胞质尾部中的高度保守序列表明与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FACS和下一代测序，以定量确定 FcRn结构对IgG再循环的影响。我们将利用能够用于Illumina的慢病毒FcRn表达文库， 深度测序该文库包括TM或胞质FcRn结构域中的非偏倚突变， 对于每个天然残基引入19个不同的氨基酸取代（总共67个残基）-而另外 保持其余蛋白质为野生型。我们将有几千个细胞表达每一个突变， 与WT相比，突变体再循环IgG的能力的比较将确定突变体再循环IgG的显著性。 突变我们将使用这种新的方法来测试假设驱动的突变，以描绘结构特征， TM区有助于有效内体再循环和胞吞转运。膜定位 蛋白质可受到诸如TM长度的性质的影响。我们还将确定FcRn是否使用不同的 二聚化基序，以在功能二聚体之间切换，如已显示的其他蛋白质。我们将使用这个 描述高度保守的胞质结构域中的单个残基、基序或连接的技术， 协助有效的内体再循环和胞吞转运。研究表明，一种亲膜两亲螺旋 可以感测/引起曲率。还有几个小的可能基序（YXXΦ，酸性二亮氨酸，CaM结合， 磷酸化位点），其将通过系统性螺旋插入/缺失/加扰来探测。我们将执行 这种跨细胞类型的慢病毒筛选;这些结果的比较将提供关于一般性的信息， 或者突变命中的特异性。无论是假设驱动的突变，还是来自非- 偏向性突变将用我们实验室中使用的再循环和转胞吞测定法进一步表征。高- 所制作的分辨率图将为实地提供广泛的实用性，作为一个模板， 膜结构/功能的关系，在任何蛋白质中的功能是服从基于FACS的 单细胞读出和分选。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训练 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。