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1.
Nat Methods ; 10(3): 259-64, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23396283

ABSTRACT

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. 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 receptors, channels and enzymes involved in many established and previously unreported interactions. Prominent among the newly identified interacting proteins were enzymes that regulate sugars, glycerolipids and cholesterol itself as well as proteins 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.


Subject(s)
Carrier Proteins/metabolism , Cholesterol/metabolism , Proteome/metabolism , Proteomics/methods , Binding, Competitive , Cell Culture Techniques , Cholesterol/biosynthesis , Cholesterol/chemistry , Cholesterol/pharmacology , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Molecular Probes/chemistry , Protein Binding , Protein Interaction Mapping , Stereoisomerism , Sterols/chemistry , Tandem Mass Spectrometry , Ultraviolet Rays
2.
Proc Natl Acad Sci U S A ; 109(13): 4768-73, 2012 Mar 27.
Article in English | MEDLINE | ID: mdl-22411830

ABSTRACT

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-E-specific 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.


Subject(s)
Axons/pathology , Axons/physiology , Chondroitin Sulfate Proteoglycans/immunology , Epitopes/immunology , Nerve Regeneration/physiology , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Axons/drug effects , Carbohydrate Conformation , Chickens , Chondroitin Sulfate Proteoglycans/chemistry , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/immunology , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Growth Cones/drug effects , Growth Cones/metabolism , Growth Cones/pathology , Mice , Neurites/enzymology , Receptor-Like Protein Tyrosine Phosphatases, Class 2/metabolism , Signal Transduction/drug effects
3.
J Biol Chem ; 287(11): 7990-8000, 2012 Mar 09.
Article in English | MEDLINE | ID: mdl-22194604

ABSTRACT

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. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.


Subject(s)
Acyltransferases/metabolism , Bacterial Proteins/metabolism , Glycolipids/biosynthesis , Mycobacterium tuberculosis/metabolism , Virulence Factors/biosynthesis , Acylation/physiology , Acyltransferases/antagonists & inhibitors , Acyltransferases/genetics , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Biological Transport, Active/drug effects , Biological Transport, Active/physiology , Enzyme Inhibitors/pharmacology , Glycolipids/genetics , Lactones/pharmacology , Mycobacterium tuberculosis/genetics , Orlistat , Virulence Factors/genetics
4.
Nat Methods ; 9(1): 84-9, 2011 Nov 06.
Article in English | MEDLINE | ID: mdl-22056678

ABSTRACT

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. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins 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.


Subject(s)
Cysteine/metabolism , Fatty Acids, Unsaturated/metabolism , Lipoylation , Palmitic Acid/metabolism , Animals , Lipase/antagonists & inhibitors , Lipoylation/drug effects , Mass Spectrometry , Mice , Organophosphonates/pharmacology , Protein Processing, Post-Translational , Serine Endopeptidases/metabolism , Thiolester Hydrolases/metabolism
5.
Proc Natl Acad Sci U S A ; 108(24): 9747-52, 2011 Jun 14.
Article in English | MEDLINE | ID: mdl-21628576

ABSTRACT

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.


Subject(s)
Glycosaminoglycans/chemistry , Microarray Analysis/methods , Models, Molecular , Proteins/chemistry , Amino Acid Sequence , Animals , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Antigens, Protozoan/metabolism , Binding Sites/genetics , Carbohydrate Sequence , Carbohydrates/analysis , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/metabolism , Computer Simulation , Fibroblast Growth Factor 2/chemistry , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Glycosaminoglycans/metabolism , Molecular Sequence Data , Oligosaccharides/chemistry , Oligosaccharides/metabolism , PC12 Cells , Protein Binding , Protein Structure, Tertiary , Proteins/genetics , Proteins/metabolism , Rats , Receptor, Fibroblast Growth Factor, Type 1/chemistry , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Sequence Homology, Amino Acid , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
6.
FEBS Lett ; 584(16): 3557-60, 2010 Aug 20.
Article in English | MEDLINE | ID: mdl-20638387

ABSTRACT

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. The effect of pipe on Snake activation has been unknown. Here we show that Snake activation is cascade-dependent but pipe-independent. These findings support a conclusion that Snake's activation of Easter is the first spatially regulated step in the dorsoventral protease cascade.


Subject(s)
Drosophila Proteins/metabolism , Drosophila/embryology , Drosophila/metabolism , Serine Endopeptidases/metabolism , Sulfotransferases/metabolism , Animals , Animals, Genetically Modified , Body Patterning , Drosophila/genetics , Drosophila Proteins/genetics , Enzyme Activation , Female , Genes, Insect , Mutation , Serine Endopeptidases/genetics , Signal Transduction , Sulfotransferases/genetics
7.
J Am Chem Soc ; 132(10): 3264-5, 2010 Mar 17.
Article in English | MEDLINE | ID: mdl-20178358

ABSTRACT

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. We show that these probes accurately report on the sn-1 and sn-2 substrate specificities of phospholipases in cell and tissue proteomes, including the sn-1-selective phospholipase DDHD1 and a calcium-dependent transacylase activity implicated in endocannabinoid biosynthesis. We anticipate that these phospholipase-directed ABPP probes will facilitate the discovery of new lipid-metabolizing enzymes and provide valuable insights into their substrate preferences.


Subject(s)
Alkynes/chemistry , Organophosphonates/chemistry , Phospholipases/analysis , Proteome/analysis , Alkynes/metabolism , Animals , Mice , Organophosphonates/metabolism , Phospholipases/classification , Phospholipases/metabolism , Proteome/metabolism , Rats , Substrate Specificity
8.
Chembiochem ; 10(8): 1340-3, 2009 May 25.
Article in English | MEDLINE | ID: mdl-19402090

ABSTRACT

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.


Subject(s)
Complement C3/chemistry , Complement C4b/chemistry , Sulfhydryl Compounds/chemistry , alpha-Macroglobulins/chemistry , Biotin/chemistry , Complement C3/immunology , Complement C4b/immunology , Fluorescent Dyes/chemistry , Peptides/chemistry , Protein Engineering , alpha-Macroglobulins/immunology
9.
J Neurochem ; 103(2): 749-60, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17680989

ABSTRACT

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. CS-E polysaccharide, but not CS-A, -C or -D polysaccharide, facilitated the neurite outgrowth of dopaminergic neurons at CS concentrations within the physiological range. The stimulatory effect of CS-E polysaccharide on neurite outgrowth was completely abolished by its digestion into disaccharide units with chondroitinase ABC. Similarly to CS-E polysaccharide, a synthetic tetrasaccharide displaying only the CS-E sulfation motif stimulated the neurite outgrowth of dopaminergic neurons, whereas a CS-E disaccharide or unsulfated tetrasaccharide had no effect. Analysis of the molecular mechanisms revealed that the action of the CS-E tetrasaccharide was mediated through midkine-pleiotrophin/protein tyrosine phosphatase zeta and brain-derived neurotrophic factor/tyrosine kinase B receptor pathways, followed by activation of the two intracellular phospholipase C (PLC) signaling cascades: PLC/protein kinase C and PLC/inositol 1,4,5-triphosphate/inositol 1,4,5-triphosphate receptor signaling leading to intracellular Ca(2+) concentration-dependent activation of Ca(2+)/calmodulin-dependent kinase II and calcineurin. These results indicate that a specific sulfation motif, in particular the CS-E tetrasaccharide unit, represents a key structural determinant for activation of midkine, pleiotrophin and brain-derived neurotrophic factor-mediated signaling, and is required for the neuritogenic activity of CS in dopaminergic neurons.


Subject(s)
Chondroitin Sulfates/chemistry , Chondroitin Sulfates/pharmacology , Dopamine/physiology , Neurites/drug effects , Neurons/physiology , Type C Phospholipases/physiology , Animals , Brain-Derived Neurotrophic Factor/physiology , Carrier Proteins/metabolism , Cells, Cultured , Cytokines/metabolism , Enzyme Activation/drug effects , Female , Fluorescent Antibody Technique , Immunohistochemistry , Mesencephalon/cytology , Mesencephalon/drug effects , Mesencephalon/growth & development , Neurites/ultrastructure , Neurons/ultrastructure , Oligosaccharides/chemistry , Oligosaccharides/pharmacology , Pregnancy , Rats , Rats, Sprague-Dawley , Receptor, trkB/metabolism , Signal Transduction/drug effects , Structure-Activity Relationship
10.
Nat Chem Biol ; 2(9): 467-73, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16878128

ABSTRACT

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. These results provide both fundamental insights into the role of sulfation and direct evidence for a 'sulfation code' whereby glycosaminoglycans encode functional information in a sequence-specific manner analogous to that of DNA, RNA and proteins.


Subject(s)
Glycosaminoglycans , Sulfates , Animals , Cell Enlargement/drug effects , Cells, Cultured , Chondroitin Sulfates/chemical synthesis , Chondroitin Sulfates/chemistry , Chondroitin Sulfates/pharmacology , Glycosaminoglycans/chemical synthesis , Glycosaminoglycans/chemistry , Glycosaminoglycans/pharmacology , Hippocampus/cytology , Hippocampus/embryology , Models, Molecular , Molecular Structure , Neurites/drug effects , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Sulfates/chemical synthesis , Sulfates/chemistry , Sulfates/pharmacology
11.
J Am Chem Soc ; 128(24): 7740-1, 2006 Jun 21.
Article in English | MEDLINE | ID: mdl-16771479

ABSTRACT

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. We anticipate that carbohydrate microarrays will accelerate our understanding of glycosaminoglycan-protein interactions and the role of sulfation in modulating physiological and disease states.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Chondroitin Sulfates/metabolism , Protein Array Analysis , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Binding Sites , Cell Membrane/metabolism , Humans , Immunoblotting , U937 Cells/drug effects
12.
J Am Chem Soc ; 126(25): 7736-7, 2004 Jun 30.
Article in English | MEDLINE | ID: mdl-15212495

ABSTRACT

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.


Subject(s)
Chondroitin Sulfates/pharmacology , Neurons/drug effects , Animals , Cells, Cultured , Chondroitin Sulfates/chemical synthesis , Chondroitin Sulfates/chemistry , Hippocampus , Microscopy, Fluorescence , Neurons/physiology , Oligosaccharides/chemical synthesis , Oligosaccharides/metabolism
13.
Chirality ; 14(4): 340-6, 2002 May 05.
Article in English | MEDLINE | ID: mdl-11968076

ABSTRACT

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. The synthesis also establishes the absolute configuration of the enantiomers. Only one of the enantiomers binds to opsin, thus showing the steric restrictions regarding the middle polyene moiety of the retinoid molecule; this is in sharp contrast to the known leniency of the ring moiety binding site of retinoids. However, although one enantiomer is incorporated into the pigment, the circular dichroic spectrum of the pigment incorporating the bound enantiomer yields only a very weak Cotton effect, showing that, once incorporated, the bicyclo[5.1.0]octanyl chromophore is flattened by the opsin binding site. The titled retinoid was synthesized for study of the absolute conformation of the retinal pigment in rhodopsin.


Subject(s)
Bridged Bicyclo Compounds/chemical synthesis , Bridged Bicyclo Compounds/metabolism , Retinaldehyde/chemical synthesis , Retinaldehyde/metabolism , Rod Opsins/metabolism , Animals , Binding Sites , Bridged Bicyclo Compounds/chemistry , Cattle , Circular Dichroism , In Vitro Techniques , Magnetic Resonance Spectroscopy , Retinaldehyde/chemistry , Stereoisomerism
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