Photocrosslinking probes to discover glycan-dependent interactions

点击翻译按钮获取中文摘要

The goal of this project is to develop accessible and effective methods to discover the interaction partners of proteins that are modified with O-linked beta-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a common post- translational modification of intracellular proteins in metazoa and higher plants. Hundreds of O-GlcNAc- modified nuclear and cytoplasmic proteins have been identified. Although O-GlcNAc is both abundant and essential, little is known about how it affects the function of modified proteins. The central hypothesis of the proposed work is that O-GlcNAc-modified proteins are surrounded by a different set of molecules than the unmodified proteins. Obtaining information about the interaction partners of O-GlcNAc-modified proteins will provide new insight into O-GlcNAc function. To identify O-GlcNAc interaction partners, we will rely on a photocrosslinking approach to convert low-affinity glycan-dependent interactions to high-affinity covalent complexes that can be purified and characterized by mass spectrometry. Two complementary approaches will be pursued: an in-lysate photocrosslinking approach (Aim 1) and an in-cell photocrosslinking approach (Aim 2). In the first aim, a panel of photocrosslinking glycopeptide probes will be prepared. These molecules will be crosslinked to proteins in lysate. The crosslinked complexes will be isolated and the interaction partners will be identified by mass spectrometry. The outcome of this aim will be an in-lysate photocrosslinking method that has the following features: (a) probe molecules produced by routine, simple methods, (b) probe molecules crosslinked efficiently and specifically to interacting proteins, (c) LC-MS/MS delivering a high yield of tryptic peptides from a specific subset of proteins, and (d) candidate interacting proteins confirmed by in vitro and cell-based assays. In the second aim, a photocrosslinking functional group will be incorporated onto O-GlcNAc residues in living cells. Subsequent UV irradiation will result in covalent crosslinking of O-GlcNAc-associated interactions in their native cellular context. As in Aim 1, the crosslinked complexes will be isolated and the interaction partners will be identified by mass spectrometry. The basic features of this in-cell photocrosslinking method have already been reported; the aim of this proposal is to make the method more effective and powerful by (a) engineering cells to produce more O-GlcNDAz, (b) purifying crosslinked material more efficiently and stringently, and (c) improving confidence in candidate hit identification. The reagents and methods developed will be shared with other research groups to enable study of a wide variety of O-GlcNAc- modified proteins with diverse biological functions. The proposed work places a high priority on approaches that are simple to implement and make use of “off-the-shelf” reagents and procedures. Making these methods available to the broad biomedical community is significant because dysregulation of O-GlcNAc is associated with multiple disease states including type II diabetes, cardiovascular disease, Alzheimer's disease, and several cancers.