Protein Glycosylation: Mechanisms and Consequences

用一种称为O-GlcNAc的特殊糖对蛋白质进行化学修饰是包括人类在内的多细胞生物中普遍存在的过程。超过一千种细胞内蛋白质被可逆地O-GlcNAc酰化，这种糖的存在影响了从我们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，尽管其流行，蛋白质O-GlcNAc酰化的调节及其影响细胞功能的机制在详细的分子水平上仍然知之甚少。因此，我们的研究计划的总体目标是双重的。 首先，我们的目标是确定称为O-GlcNAc转移酶（或简称OGT）的酶如何选择它用O-GlcNAc修饰的特定蛋白质。除了发生O-GlcNAc化学转移的催化结构域之外，OGT还具有称为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，从而通过相邻的催化结构域指导它们的修饰。因此，我们将使用各种生物化学筛选来搜索和鉴定结合TPR结构域的蛋白质。一旦确定，我们将使用强大的NMR光谱技术作为“分子显微镜”来定义支持其识别的底物蛋白质的性质，并随后通过OGT进行O-GlcNAc修饰。 其次，我们将研究O-GlcNAc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的作用可能是环境依赖性的。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的相互作用或阻断替代的调节化学修饰，如磷酸化。因此，我们将使用NMR光谱等技术来确定O-GlcNAc如何影响参与基因表达，信号传导和细胞组织的代表性蛋白质组的分子结构，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 O-GlcNAc酰化，以及这种修饰如何影响它们的功能。这一基本知识应提供科学界急需的分子水平上的理解所发挥的作用，在许多正常和异常的细胞过程中的O-GlcNAcylation。我们的研究还将为从生物物理学到细胞生物学等领域的研究生和博士后研究员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.