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Chemical Neurobiology
Summary: Linda Hsieh-Wilson's research combines organic chemistry and neurobiology to study the roles of carbohydrates and associated proteins in transcription, cell signaling, and neuronal regeneration. Her lab develops chemical tools for the proteome-wide identification of glycoproteins, for imaging glycoproteins in cells, and for monitoring altered glycosylation states of proteins.
Our research is focused on understanding how carbohydrates regulate the structure and function of proteins in the central nervous system. Protein glycosylation has been shown to be ubiquitous, with carbohydrates implicated in diverse functions, ranging from learning and memory to brain development and spinal cord regeneration. A central challenge is to understand the structure-activity relationships of carbohydrates and their roles in various biological contexts; however, traditional approaches of biochemistry and genetics are not well suited to this task. Biochemical methods provide mixtures of heterogeneously glycosylated proteins, while genetic perturbation of a glycosyltransferase gene can alter various carbohydrate structures on multiple different glycoproteins.
To address this challenge, our laboratory is developing chemical tools to study carbohydrates and their physiological roles in the brain. Our research focuses on three carbohydrate modifications: (1) a dynamic form of glycosylation called O-GlcNAc; (2) the glycosaminoglycan class of sulfated polysaccharides; and (3) the sugar fucose, a terminal modification found on glycoproteins. Our work has demonstrated that a wide variety of neuronal proteins undergo these 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 developed a chemoenzymatic strategy to detect and identify O-GlcNAc–glycosylated proteins rapidly. Our approach employs an engineered galactosyltransferase to transfer a synthetic ketone- or azide-containing substrate to O-GlcNAc–modified proteins. Once installed, the ketone or azide moiety can be selectively reacted with radiolabels, affinity tags, or chemiluminescent probes. This 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, facilitates studies of the interplay between phosphorylation and glycosylation, and allows for cellular imaging of the glycoproteins. We have extended the approach to enable the first direct proteome-wide analysis of O-GlcNAc–glycosylated proteins. We identified more than 200 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.
An emerging theme from our studies is that O-GlcNAc glycosylation may play critical roles in the regulation of gene expression. We identified the O-GlcNAc modification on numerous transcription factors and transcriptional regulatory proteins (e.g., CREB, ATF-2, Sox2, ΔFosB, CBP) involved in regulating processes such as drug addiction, hormone receptor activation, and memory consolidation. We also discovered that the modification extends beyond transcription factors to new classes of proteins, including transcriptional corepressors, coactivators, and chromatin-remodeling enzymes. These findings suggest that O-GlcNAc glycosylation may serve as a general mechanism for the control of transcription. Supporting this notion, our studies indicate that O-GlcNAc glycosylation of the transcription factor CREB (cyclic AMP response element–binding protein) represses gene expression and contributes to pancreatic β-cell death by impairing association of the transcription factor with TAFII130, a component of the transcriptional machinery.
Our studies also reveal that O-GlcNAc glycosylation represents a novel form of communication in the brain. Recently, we developed chemical methods to study the dynamics of O-GlcNAc glycosylation on a proteome-wide scale. We found that O-GlcNAc glycosylation is induced by neuronal activity, such as KCl depolarization of neurons or kainic acid–induced excitatory stimulation in vivo. Activation of O-GlcNAc glycosylation requires calcium influx into the cell and calcium- and calmodulin-dependent protein kinases. Current studies in our laboratory seek to determine how O-GlcNAc glycosylation is dynamically regulated at synapses and its implications for dendritic protein synthesis and synaptic plasticity.
Glycosaminoglycans
Understanding the barriers that prevent nerve cell regeneration is critical to the development of new treatments for neuronal injury and neurodegenerative diseases. Our studies focus on chondroitin sulfate (CS), a key polysaccharide that plays important roles during brain development and blocks axon regrowth after central nervous system injury. CS polysaccharides display diverse sulfation patterns that are spatiotemporally regulated in vivo. We are interested in understanding how specific sulfation motifs modulate the activity of proteins and contribute to the regulation of neuronal growth and regeneration. Toward this end, we have developed chemical methods to synthesize CS glycosaminoglycans, in which sulfate groups are installed at precise positions along the carbohydrate chain.
Our studies have revealed that a specific sequence, called CS-E, exerts both growth-promoting and growth-inhibiting effects on neurite outgrowth in vitro. These effects are context-dependent and appear to involve the interaction of CS-E with specific protein partners. For instance, we have found that CS-E interacts with the neurotrophin family of growth factors, as well as myelin-associated inhibitory proteins. Blocking the CS-E motif with a monoclonal antibody reversed the neurite inhibition mediated by CS proteoglycans and stimulated optic nerve regeneration in vivo. Thus, using antibodies or other reagents to target specific CS epitopes may provide a promising new strategy to promote axon regeneration and neural plasticity after injury.
More broadly, our results provide support for the concept of a "sulfation code," whereby the sulfation patterns of glycosaminoglycans encode functional information in a sequence-specific manner analogous to DNA, RNA, and proteins. We are investigating the functional roles and spatiotemporal expression of CS-E and other sulfation sequences in the brain. In addition, we are exploring the therapeutic potential of the CS-E antibody to promote axon regeneration after spinal cord 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 chemical tools to identify proteins associated with this sugar to understand its functional roles in the nervous system. Our studies have demonstrated that fucosylated proteins are enriched at neuronal synapses and are prevalent in both the developing and adult rat brain. We identified more than 25 fucosylated proteins, including cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle–associated proteins, and mitochondrial proteins. Two of the major fucosylated proteins in the adult brain are synapsin Ia and Ib, which play important roles in the regulation of neurotransmitter release and neuronal morphology. Fucosylation protects synapsin Ia and Ib from cellular degradation by the protease calpain. 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 key 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 June 11, 2009
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