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Chemical Neurobiology
Summary: Linda Hsieh-Wilson’s research combines organic chemistry and neurobiology to study the roles of carbohydrates and their associated proteins in transcription, neuronal signaling, and brain development. Her lab develops chemical tools for the proteome-wide identification of glycoproteins, for visualizing glycoproteins within cells, and for monitoring altered glycosylation states of proteins.
Given that the complexity of higher organisms is encoded in a remarkably small number of genes, increasing attention has focused on other mechanisms to account for the diversity of biological systems. Protein post-translational modifications represent one such mechanism—the covalent attachment of phosphate, carbohydrate, lipid, and other groups to proteins expands their capabilities and provides exquisite spatiotemporal control over protein function. We study protein glycosylation and seek to understand how specific carbohydrates regulate the structure and function of neuronal proteins. Our work has demonstrated that a wide variety of neuronal proteins undergo diverse carbohydrate modifications, and it has defined exciting new roles for protein glycosylation in the brain.
Dynamic O-GlcNAc Glycosylation
O-GlcNAc glycosylation, the addition of β-N-acetylglucosamine to serine or threonine residues of proteins, is a dynamic, intracellular modification that shares features with protein phosphorylation. Recent studies have suggested diverse roles for the O-GlcNAc modification, ranging from nutrient sensing and proteasomal regulation to gene silencing. Moreover, perturbations in O-GlcNAc levels have been associated with diseases such as cancer, Alzheimer's disease, and diabetes.
We have discovered that the transcription factor cyclic AMP response element–binding protein (CREB) is O-GlcNAc glycosylated in vivo. Our studies reveal that glycosylation impairs the association of CREB with TAFII130, a component of the transcriptional machinery, and represses the transcriptional activity of CREB. As CREB regulates key physiological processes, such as glucose homeostasis, neuronal survival, and long-term memory storage, these findings provide an opportunity to understand the roles of the O-GlcNAc modification in specific cellular contexts. Toward this end, we have shown that glycosylation represses the activity of CREB in insulin-producing β cells and down-regulates IRS-2, a gene critical for β-cell survival. Glucosamine-induced β-cell death is blocked by mutation of the glycosylation sites on CREB or by depletion of O-GlcNAc transferase through RNA interference (RNAi). Thus, O-GlcNAc glycosylation is a novel mechanism for CREB regulation that may have important implications for glucose homeostasis and diabetes. On the basis of our results, we have proposed that chronic hyperglycemia leads to hyperglycosylation of CREB, thereby disrupting IRS-2 expression and contributing to pancreatic β-cell death.
Another component of our program is the development of chemical tools to accelerate the detection and study of O-GlcNAc modifications. We employ an engineered galactosyltransferase to transfer a synthetic ketone-containing substrate to O-GlcNAc–modified proteins. Once installed, the ketone can be elaborated with radiolabels, affinity tags, or chemiluminescent probes. Our approach overcomes a key challenge to the study of O-GlcNAc: detection of this low-abundance, labile modification. It also permits any protein to be readily interrogated for the modification in vivo and facilitates studies of the interplay between phosphorylation and glycosylation. We have extended the approach to enable the first direct proteome-wide analysis of O-GlcNAc–glycosylated proteins. We identified more than 40 new O-GlcNAc–glycosylated proteins from the mammalian brain, including proteins involved in gene expression, neuronal signaling, and synaptic plasticity. These results significantly expand the number of proteins known to possess the modification and suggest that O-GlcNAc glycosylation plays broader roles in the brain than previously appreciated.
In the past year, we have developed a chemical method to study the dynamics of O-GlcNAc glycosylation in vivo on a proteome-wide scale. Our studies indicate that the O-GlcNAc modification is reversible and dynamically induced in the brain by robust excitatory stimulation. We identified several neuronal proteins that undergo changes in O-GlcNAc glycosylation level upon treatment of rats with the glutamate receptor agonist kainic acid. Our data indicate that O-GlcNAc is modulated by specific signaling pathways and may play a role in neuronal communication, reinforcing the notion that O-GlcNAc glycosylation represents a key, regulatory modification in the brain. Current studies in our laboratory seek to identify the signals and pathways that activate O-GlcNAc in neurons, both on a proteome-wide scale and on individual proteins such as CREB. A key obstacle in reaching this goal is the difficulty of monitoring glycosylation levels on specific proteins of interest in response to a host of stimuli. We have recently developed a new method for solving this problem.
Glycosaminoglycans
Glycosaminoglycans are a family of sulfated polysaccharides involved in neuronal development, viral invasion, and spinal cord injury. Their structures display diverse patterns of sulfation, which are tightly regulated in vivo. We are interested in understanding how specific sulfation motifs modulate the activities of proteins and contribute to the regulation of neuronal growth and development. Toward this end, we are developing chemical methods to synthesize chondroitin sulfate (CS) glycosaminoglycans, in which sulfate groups are installed at precise positions along the carbohydrate chain. We used these defined structures to demonstrate that a specific sulfation motif, CS-E, functions as a molecular recognition element for growth factors and thereby modulates the outgrowth of neurons. These results provide support for the concept of a "sulfation code," whereby the sulfation patterns of glycosaminoglycans may encode functional information in a sequence-specific manner analogous to DNA, RNA, and proteins. We are investigating the spatiotemporal expression of CS-E in the brain and the functional roles of other sulfation sequences. In addition, we are pursuing x-ray crystal structures of various growth factors and their receptors complexed to defined CS molecules.
In the past year, we have developed novel glycosaminoglycan mimetics to explore how the macromolecular structure of CS directs its activity and to manipulate CS function in vivo. Our approach greatly simplifies the chemical synthesis of glycosaminoglycans, providing synthetically accessible, bioactive structures of programmable sulfation sequence. These glycopolymers highlight the importance of multivalency in amplifying CS activity and reveal an unexpected tolerance of CS-binding proteins for unnatural polymeric architectures. We are generating various glycopolymers with specific sulfation codes to probe the roles of CS in neural development and spinal cord injury. Finally, we are examining whether our synthetic compounds have growth-promoting activities in vivo as a potential treatment for neuronal injury.
Protein Fucosylation
The attachment of the sugar L-fucose to proteins has been implicated in cognitive processes such as learning and memory storage. We are developing proteomics and chemical tools to identify proteins associated with this sugar to understand its functional roles in the nervous system. We have found that fucosylated proteins are enriched at neuronal synapses and are prevalent in the developing and adult rat brain. Our studies have identified synapsin Ia and Ib, proteins important for the regulation of neurotransmitter release and neuronal morphology, as two of the major fucosylated proteins in the adult brain. Fucosylation of synapsin Ia and Ib protects them from cellular degradation by the protease calpain. Furthermore, inhibition of fucosylation on synapsin and other proteins impairs neurite outgrowth and delays synapse formation. We have also found that fucose-binding lectins exist in neurons and participate in a pathway that modulates neurite outgrowth. Together, our studies identify important roles for fucosyl sugars in the regulation of neuronal proteins and morphological changes that may underlie synaptic plasticity.
This work is also supported by grants from the National Institutes of Health, the American Cancer Society, the Tobacco-Related Disease Research Program, and the National Science Foundation.
Last updated: April 23, 2008
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