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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

基本信息

批准号：
9911979
负责人：
Jamie S LeBarron
金额：
$ 6.74万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9911979
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 Epithelium 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

项目摘要

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.

项目总结