Protein Glycosylation: Mechanisms and Consequences

在包括人类在内的多细胞生物中，用一种叫做O-GlcNAc的特殊糖对蛋白质进行化学修饰是一个普遍存在的过程。超过一千种细胞内蛋白质被可逆地o - glcn酰化，这种糖的存在影响了从遗传密码的表达到细胞信号传导和各种形式的应激反应的各种生化过程。然而，尽管o - glcn酰化很普遍，但在分子水平上对蛋白o - glcn酰化的调控及其影响细胞功能的机制仍知之甚少。因此，我们研究项目的总体目标是双重的。首先，我们的目标是确定O-GlcNAc转移酶（简称OGT）如何选择它用O-GlcNAc修饰的特定蛋白质。除了O-GlcNAc发生化学转移的催化结构域外，OGT还有一个被称为TPR结构域的长尾。假设TPR结构域是识别（结合）某些底物蛋白的支架，从而通过邻近的催化结构域指导它们的修饰。因此，我们将使用各种生化筛选来搜索和识别结合TPR结构域的蛋白质。一旦确定，我们将使用强大的核磁共振波谱技术作为“分子显微镜”来定义底物蛋白的特性，支持它们的识别和随后的OGT修饰O-GlcNAc。其次，我们将研究o - glcn酰化对蛋白质功能的影响。考虑到与这种修饰相关的多种细胞过程，O-GlcNAc的作用可能与环境有关。在某些情况下，O-GlcNAc的存在似乎可以防止不需要的蛋白质聚集，而在其他情况下，它似乎可以促进或破坏与伴侣细胞蛋白质的相互作用，或阻断其他调节化学修饰，如磷酸化。因此，我们将使用核磁共振波谱等技术来确定O-GlcNAc如何影响参与基因表达、信号传导和细胞组织的一组具有代表性的蛋白质的分子结构、运动和相互作用。我们的研究结果将有助于解释哪些蛋白质被OGT酰基化，以及这种修饰如何影响它们的功能。这一基础知识将为科学界提供一个非常需要的分子水平的理解，即o - glcn酰化在许多正常和异常的细胞过程中所起的作用。我们的研究还将为从生物物理学到细胞生物学的研究生和博士后提供良好的培训和职业发展机会。

The chemical modification of proteins with a specialized sugar called O-GlcNAc is a ubiquitous process in multicellular organisms, including humans. Over a thousand intracellular proteins are reversibly O-GlcNAcylated, and the presence of this sugar influences a diverse range of biochemical processes spanning from the expression of our genetic code to cellular signaling and various forms of stress response. However, despite its prevalence, the regulation of protein O-GlcNAcylation and the mechanisms by which it impacts cellular functions remain poorly understood at a detailed molecular level. Accordingly, the overall goals of our research program are two-fold.First, we aim to determine how the enzyme called O-GlcNAc transferase (or simply OGT) selects the specific proteins that it modifies with O-GlcNAc. In addition to the catalytic domain where the chemical transfer of O-GlcNAc occurs, OGT has a long tail called a TPR domain. It is hypothesized that the TPR domain is a scaffold that recognizes (binds) certain substrate proteins, and thereby directs their modification by the adjacent catalytic domain. Thus we will use a variety of biochemical screens to search for and identify proteins that bind the TPR domain. Once identified, we will use the powerful technique of NMR spectroscopy as a "molecular microscope" to define the properties of the substrate proteins underpinning their recognition and subsequent O-GlcNAc modification by OGT.Second, we will investigate the consequences of O-GlcNAcylation on protein function. Given the diverse cellular processes associated with this modification, it is likely that the effects of O-GlcNAc are context-dependent. In some cases, the presence of O-GlcNAc appears to prevent unwanted protein aggregation, whereas in other cases, it seems to promote or disrupt interactions with partner cellular proteins or block alternative regulatory chemical modifications such as phosphorylation. Therefore, we will use techniques such as NMR spectroscopy to determine how O-GlcNAc influences the molecular structure, motions and interactions of a representative set of proteins involved in gene expression, signaling and cellular organization. The results of our studies will help explain which proteins are O-GlcNAcylated by OGT and how this modification impacts their function. This fundamental knowledge should provide the scientific community with a much needed molecular-level understanding of the roles played by O-GlcNAcylation in many normal and aberrant cellular processes. Our research will also offer excellent training and career advancement opportunities for graduate students and postdoctoral fellows in areas ranging from biophysics to cellular biology.