No abstract

The reversible thioester linkage of palmitic acid on cysteines is known as protein S-palmitoylation, which facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analogue 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling of cells (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 show rapid turnover. Treatment with a serine lipase-selective inhibitor identified a special pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncogenes and other proteins linked to aberrant cell growth, migration, and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.

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 engaging in specific interactions with proteins. The full complement of sterol-binding proteins that exist in mammalian cells, however, remains unknown. Here, we describe a chemoproteomic strategy that uses clickable, photoreactive sterol probes in combination with quantitative mass spectrometry to globally map cholesterol-protein interactions directly in living cells. We identified over 250 cholesterol-binding proteins, including many established and previously unreported interactions with receptors, channels, and enzymes. Prominent among the newly identified interactions were enzymes that regulate sugars, glycerolipids, and cholesterol itself, as well as those involved in vesicular transport and protein glycosylation and degradation, pointing to key nodes in biochemical pathways that may couple sterol concentrations to the control of other metabolites and protein localization and modification.

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. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein tyrosine phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-Especific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury.

General MethodsUnless stated otherwise, reactions were performed in flame-dried glassware under a nitrogen or an argon environment, using freshly distilled solvents. All other commercially obtained reagents were used as received. Thinlayer chromatography (TLC) was performed using E. Merck silica gel 60 F254 precoated plates (0.25 mm).Visualization of the developed chromatogram was performed by fluorescence quenching, cerium ammonium molybdate stain, or ninhydrin stain as necessary. ICN silica gel (particle size 0.032 -0.063 mm) was used for flash chromatography. Gel filtration chromatography (Sephadex G-10 and G-25 ultrafine) was used in order to achieve purification of the final products.

Background: Sulfolipid-1 (SL-1) is a Mycobacterium tuberculosis outer membrane lipid whose biosynthesis is not fully understood.Results: Chp1 catalyzes two acyl transfer reactions to form SL-1. Sap modulates SL-1 levels and transmembrane transport.Conclusion: The activities of Chp1 and Sap complete the SL-1 pathway.Significance: Lipid biosynthesis and transport are coupled at the membrane interface by multiple proteins that may regulate substrate specificity and flux.

Conjugation of CS Oligosaccharides to 1,2-(Bisaminooxy)ethane for Microarray ProductionOzonolysis of the anomeric allyl group and linkage of CS compounds 1-4 1,2 to 1,2-(bisaminooxy)ethane 3 proceeded as follows: oligosaccharide (0.51 µmol) was dissolved in MeOH (500 µL) and cooled to -78 °C. O 3 was bubbled through the reaction until a blue color persisted (1 min). The reaction was then purged with N 2 until colorless, quenched with Ph 3 P beads (3 mg), and gradually warmed to rt over 12 h. It was filtered and the product concentrated to afford the desired aldehyde as a white solid. The aldehyde (0.51 µmol) was then reacted for 14 h at rt with 1,2-(bisaminooxy)ethane hydrochloride (1.4 mg, 15 µmol) that had been dissolved in H 2 O (100 µL) and pH adjusted to 5.0 with 1 M NaOH. The resulting oxime product was purified using a SepPak C18 column (500 mg, H 2 O) and Sephadex G-10 (CS-E The relative concentrations of the aminooxy oligosaccharides were calibrated to one another using the carbazole assay for uronic acid residues. 4 Briefly, the acid borate reagent (1.5 mL of 0.80 g sodium tetraborate, 16.6 mL H 2 O, and 83.3 mL H 2 SO 4 ) was added to 20-mL glass vials with Teflon caps. The Carbohydrate MicroarraysSolutions of the aminooxy oligosaccharides (in 300 mM NaH 2 PO 4 , pH 5.0, 10 µL/well in a 384-well plate) were arrayed on Hydrogel Aldehyde slides (NoAb Biodiscoveries) by using a Microgrid II arrayer (Biorobotics) to deliver sub-nanoliter volumes at rt and 50% humidity. Concentrations of carbohydrates ranged from 0 -500 µM. The resulting arrays were incubated in a 70% humidity chamber at rt for 12 h and then stored in a low humidity, dust-free dessicator. The pH and reaction time were Non-specific attachment of CS oligosaccharides lacking the aminooxy linker (e.g., compounds 1-4) was not observed. Prior to use, the arrays were outlined with a hydrophobic pen (Super Pap Pen, Research Products International) to create a boundary for the protein treatments and rinsed three times with H 2 O. The slides were then blocked by treatment with NaBH 4 (125 mg) in 140 mM NaCl, 2.7 mM KCl, 5.4 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 (phosphate buffered saline, PBS, 50 mL) at rt for 5 min with gentle rocking and washed five times for 3 min with PBS. For all incubations, the slides were placed in a covered pipette tip box. Human TNF-α (Peprotech), FGF-1 (R&D Systems; both reconstituted to 2 µM in 0.1% Triton X-100 in PBS), cell culture supernatant containing monoclonal anti-CS-A antibody, or cell culture supernatant containing monoclonal anti-CS-E antibody (both 1:1 in 0.1% Triton X-100 in PBS) were spotted onto the slides in 250 µL quantities, and incubated statically at rt for 2 h. The slides were then washed as previously described and incubated with the appropriate primary antibody [anti-TNF-α (Peprotech) or anti-FGF-1 (R&D Systems); 1:1000 in 0.1% Triton X-100 in PBS] for 2 h at rt with gentle rocking. Following the incubation, the slides were washed as previously described and treated in the dark at rt with a seconda...

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. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan-protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan-protein-protein interactions, as well as a molecular-level understanding of those complexes. G lycosaminoglycans (GAGs) regulate a wide range of physiological processes, including viral invasion, blood coagulation, cell growth, and spinal cord injury (1-4). Assembled from repeating disaccharide units, GAGs display diverse patterns of sulfation (SI Appendix, Fig. S1). These sulfation patterns are believed to have important functional consequences, enabling the polysaccharides to interact with a wide variety of proteins (1, 2). However, the precise sulfation motifs involved in protein recognition are understood in only a few cases (1,4,5). Moreover, studies of heparan sulfate (HS) interactions with fibroblast growth factors suggest that GAGs can assist in the assembly of multimeric protein complexes, thereby modulating signal transduction pathways (6-10). Yet, only a few such examples have been elucidated, and the extent to which other GAGs such as chondroitin sulfate (CS) engage in the formation of multimeric protein complexes remains unknown. Elucidating the interactions of specific GAG substructures with proteins and large protein-protein complexes will be critical for understanding the structure-activity relationships of GAGs and the mechanisms underlying important biological processes.Several methods have been developed to study GAG-protein interac...