Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells.

Cholesterol is an essential structural component of cellular membranes and serves as a precursor for several classes of signaling molecules. Cholesterol exerts its effects and is, itself, regulated in large part by engagement in specific interactions with proteins. The full complement of sterol-binding proteins that exist in mammalian cells, however, remains unknown.

A sulfated carbohydrate epitope inhibits axon regeneration after injury.

Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth.

Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood.

Global profiling of dynamic protein palmitoylation.

The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly.

Elucidating glycosaminoglycan-protein-protein interactions using carbohydrate microarray and computational approaches.

Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan-protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes.

Activation of Snake in a serine protease cascade that defines the dorsoventral axis is atypical and pipe-independent in Drosophila embryos.

During Drosophila embryogenesis, establishment of ventral and lateral cell fates requires spatial regulation of an extracellular serine protease cascade composed of Nudel, Gastrulation Defective (GD), Snake, and Easter. Pipe, a sulfotransferase expressed ventrally during oogenesis, sulfates secreted targets that somehow confer positive spatial input to this cascade. Nudel and GD activation are pipe-independent, while Easter activation requires pipe.

Activity-based probes that target functional subclasses of phospholipases in proteomes.

Phospholipases are a large and diverse set of enzymes that metabolize the phospholipid components of cell membranes and function in key lipid-signaling pathways. The molecular characterization of novel phospholipases would benefit from chemical probes that selectively target these enzymes on the basis of their distinct substrate specificities and catalytic properties. Here we present the synthesis and characterization of a set of activity-based protein profiling (ABPP) probes that contain key recognition and reactivity elements for targeting phospholipases of the serine hydrolase superfamily.

A chemical approach to immunoprotein engineering: chemoselective functionalization of thioester proteins in their native state.

Less than 6 feet under: Serum proteins C3, C4, and alpha(2)M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.

Activation of phospholipase C pathways by a synthetic chondroitin sulfate-E tetrasaccharide promotes neurite outgrowth of dopaminergic neurons.

In dopaminergic neurons, chondroitin sulfate (CS) proteoglycans play important roles in neuronal development and regeneration. However, due to the complexity and heterogeneity of CS, the precise structure of CS with biological activity and the molecular mechanisms underlying its influence on dopaminergic neurons are poorly understood. In this study, we investigated the ability of synthetic CS oligosaccharides and natural polysaccharides to promote the neurite outgrowth of mesencephalic dopaminergic neurons and the signaling pathways activated by CS.

Sulfation patterns of glycosaminoglycans encode molecular recognition and activity.

Although glycosaminoglycans contribute to diverse physiological processes, an understanding of their molecular mechanisms has been hampered by the inability to access homogeneous glycosaminoglycan structures. Here, we assembled well-defined chondroitin sulfate oligosaccharides using a convergent, synthetic approach that permits installation of sulfate groups at precise positions along the carbohydrate backbone. Using these defined structures, we demonstrate that specific sulfation motifs function as molecular recognition elements for growth factors and modulate neuronal growth.

Discovery of a TNF-alpha antagonist using chondroitin sulfate microarrays.

We report the first example of synthetic chondroitin sulfate (CS) microarrays to rapidly identify glycosaminoglycan-protein interactions and probe the specificity of proteins for distinct sulfation sequences. Using the microarrays, we identify a novel interaction between CS and TNF-alpha, a proinflammatory cytokine involved in rheumatoid arthritis, Crohn's disease, and psoriasis. Moreover, we demonstrate that CS-E tetrasaccharides and polysaccharides enriched in the CS-E sulfation motif can inhibit the activity of this therapeutically important cytokine.

A chondroitin sulfate small molecule that stimulates neuronal growth.

Chondroitin sulfate glycosaminoglycans are sulfated polysaccharides involved in cell division, neuronal development, and spinal cord injury. Here, we report the synthesis and identification of a chondroitin sulfate tetrasaccharide that stimulates the growth and differentiation of neurons. These studies represent the first, direct investigations into the structure-activity relationships of chondroitin sulfate using homogeneous synthetic molecules and define a tetrasaccharide as a minimal motif required for activity.

Synthesis of 11-cis-locked bicyclo[5.1.0]octanyl retinal and an enantioselective binding to bovine opsin.

Both enantiomers of 13-(E) and 13-(Z) isomers of 11-cis-locked bicyclo[5.1.0]octanyl retinal were prepared by an improved synthesis and incubated with bovine opsin.