Photocrosslinking probes to discover glycan-dependent interactions

该项目的目标是开发可访问和有效的方法来发现 用O-连接的β-N-乙酰氨基葡萄糖(O-GlcNAc)修饰的蛋白质。O-GlcNAc是一种常见的POST- 后生动物和高等植物中细胞内蛋白质的翻译修饰。数以百计的O-GlcNAc- 修饰的核蛋白和细胞质蛋白已经被鉴定出来。尽管O-GlcNAc含量丰富， 重要的是，人们对它如何影响修饰蛋白质的功能知之甚少。的核心假说 拟议的工作是O-GlcNAc修饰的蛋白质被一组不同于O-GlcNAc修饰的蛋白质的分子包围 未经修饰的蛋白质。获得O-GlcNAc修饰蛋白相互作用伙伴的信息 将为研究O-GlcNAc的功能提供新的视角。为了确定O-GlcNAc交互合作伙伴，我们将依靠 将低亲和力依赖的糖链相互作用转化为高亲和力共价作用的光交联法 可以通过质谱学进行纯化和表征的络合物。两种互补的方法将 追求：裂解物光交联法(AIM 1)和细胞内光交联法(AIM 2)。在第一个目标中，将制备一组光交联糖肽探针。这些分子将是 与裂解物中的蛋白质发生交联。交联物将被分离，相互作用伙伴将 通过质谱学进行鉴定。这一目标的结果将是一种溶剂型光交联法 它具有以下特征：(A)通过常规、简单的方法产生的探针分子，(B)探针分子 (C)高效和特异地与相互作用的蛋白质进行交联，(C)LC-MS/MS提供高产率的胰酶 来自特定蛋白质子集的多肽，以及(D)通过体外和 基于细胞的分析。第二个目的是在O-GlcNAc上引入光交联官能团 活细胞中的残留物。随后的紫外光照射将导致O-GlcNAc相关的共价交联 在它们的自然细胞环境中的相互作用。如目标1所示，将分离出交联的络合物 相互作用的伙伴将通过质谱学来确定。此单元内的基本功能 光交联法已经被报道，这项提议的目的是使该方法更 通过(A)工程细胞产生更多O-GlcNDAz，(B)提纯交联物，从而有效和强大 更高效和更严格，以及(C)提高对候选命中识别的信心。试剂和 开发的方法将与其他研究小组共享，以实现对各种O-GlcNAc- 具有多种生物学功能的修饰蛋白质。拟议的工作将方法放在高度优先的位置 这些方法易于实施和使用“现成”试剂和程序。制作这些方法 对广泛的生物医学界来说意义重大，因为O-GlcNAc的失调与 有多种疾病状态，包括II型糖尿病、心血管疾病、阿尔茨海默氏症和几种 癌症。

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.