Rhoderick E. Brown

Protein measurement using bicinchoninic acid: elimination of interfering substances

Protein quantitation based on bicinchoninic acid (BCA) is simple, sensitive, and tolerant to many detergents and substances known to interfere with the Lowry method. However, certain compounds often used during protein purification do interfere with the BCA protein assay. The response of the BCA chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by BCA. Certain substances (e.g., glucose, mercaptoethanol, and dithiothreitol) elicit a strong absorbance at 562 nm when combined with the BCA working reagent. The absorbance appears to be identical to the normal response elicited by protein. Other agents (e.g., ammonium sulfate and certain ampholytes) diminish the protein-induced color development and shift the wave-length of the color response. Both types of interference can be eliminated by selectively precipitating protein with deoxycholate and trichloroacetic acid (A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70,241-250) prior to reaction with bicinchoninic acid. The modifications described here permit quick, efficient removal of many interfering substances that are commonly utilized during protein purification.

Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol.

The effect of cholesterol on the interfacial elastic packing interactions of various molecular species of phosphatidylcholines (PCs) has been investigated by using a Langmuir-type film balance and analyzing the elastic area compressibility moduli (Cs(-1)) as a function of average cross-sectional molecular area. Emphasis was on the high surface pressure regions (pi > or = 30 mN/m) which are thought to mimic biomembrane conditions. Increasing levels of cholesterol generally caused the in-plane elasticity of the mixed monolayers to decrease. Yet, the magnitude of the cholesterol-induced changes was markedly dependent upon PC hydrocarbon structure. Among PC species with a saturated sn-1 chain but different sn-2 chain cis unsaturation levels [e.g., myristate (14:0), oleate (18:1delta9(c), linoleate (18:2delta9,12(c), arachidonate (20:4delta5,8,11,14(c), or docosahexenoate (22:6delta4,7,10,13,16,19(c)], the in-plane elasticity moduli of PC species with higher sn-2 unsaturation levels were less affected by high cholesterol mol fractions (e.g., >30 mol %) than were the more saturated PC species. The largest cholesterol-induced decreases in the in-plane elasticity were observed when both chains of PC were saturated (e.g., di-14:0 PC). When both acyl chains were identically unsaturated, the resulting PCs were 20-25% more elastic in the presence of cholesterol than when their sn-1 chains were long and saturated (e.g., palmitate). The mixing of cholesterol with PC was found to diminish the in-plane elasticity of the films beyond what was predicted from the additive behavior of the individual lipid components apportioned by mole and area fraction. Deviations from additivity were greatest for di-14:0 PC and were least for diarachidonoyl PC and didocosahexenoyl PC. In contrast to Cs(-1) analyses, sterol-induced area condensations were relatively unresponsive to subtle structural differences in the PCs at high surface pressures. Cs(-1) versus average area plots also indicated the presence of cholesterol concentration-dependent, low-pressure (<14 mN/m) phase boundaries that became more prominent as PC acyl chain unsaturation increased. Hence, area condensations measured at low surface pressures often do not accurately portray which lipid structural features are important in the lipid-sterol interactions that occur at high membrane-like surface pressures.

Involvement of the Acid Sphingomyelinase Pathway in UVA-induced Apoptosis

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10–40 μm), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20–80 μm), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.

Cholesterol Depletion Results in Site-specific Increases in Epidermal Growth Factor Receptor Phosphorylation due to Membrane Level Effects STUDIES WITH CHOLESTEROL ENANTIOMERS

In A431 cells, depletion of cholesterol with methyl-β-cyclodextrin induced an increase in both basal and epidermal growth factor (EGF)-stimulated EGF receptor phosphorylation. This increase in phosphorylation was site-specific, with significant increases occurring at Tyr845, Tyr992, and Tyr1173, but only minor changes at Tyr1045 and Tyr1068. The elevated level of receptor phosphorylation was associated with an increase in the intrinsic kinase activity of the EGF receptor kinase, possibly as a result of the cyclodextrin-induced enhancement of the phosphorylation of Tyr845, a site in the kinase activation loop known to be phosphorylated by pp60src. Cholesterol and its enantiomer (ent-cholesterol) were used to investigate the molecular basis for the modulation of EGF receptor function by cholesterol. Natural cholesterol (nat-cholesterol) was oxidized substantially more rapidly than ent-cholesterol by cholesterol oxidase, a protein that contains a specific binding site for the sterol. By contrast, the ability of nat- and ent-cholesterol to interact with sphingomyelins and phosphatidylcholine and to induce lipid condensation in a monolayer system was the same. These data suggest that, whereas cholesterol-protein interactions may be sensitive to the absolute configuration of the sterol, sterol-lipid interactions are not. nat- and ent-cholesterol were tested for their ability to physically reconstitute lipid rafts following depletion of cholesterol. nat- and ent-cholesterol reversed to the same extent the enhanced phosphorylation of the EGF receptor that occurred following removal of cholesterol. Furthermore, the enantiomers showed similar abilities to reconstitute lipid rafts in cyclodextrin-treated cells. These data suggest that cholesterol most likely affects EGF receptor function because of its physical effects on membrane properties, not through direct enantioselective interactions with the receptor.

Sphingomyelin Interfacial Behavior: The Impact of Changing Acyl Chain Composition ☆

Sphingomyelins (SMs) containing homogeneous acyl chains with 12, 14, 16, 18, 24, or 26 carbons were synthesized and characterized using an automated Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at constant temperatures between 10 degrees C and 30 degrees C. SM containing lauroyl (12:0) acyl chains displayed only liquid-expanded behavior. Increasing the length of the saturated acyl chain (e.g., 14:0, 16:0, or 18:0) resulted in liquid-expanded to condensed two-dimensional phase transitions at many temperatures in the 10-30 degrees C range. Similar behavior was observed for SMs with lignoceroyl (24:0) or (cerotoyl) 26:0 acyl chains, but isotherms showed only condensed behavior at 10 and 15 degrees C. Insights into the physico-mechanical in-plane interactions occurring within the different SM phases and accompanying changes in SM phase state were provided by analyzing the interfacial area compressibility moduli. At similar surface pressures, SM fluid phases were less compressible than those of phosphatidylcholines with similar chain structures. The area per molecule and compressibility of SM condensed phases depended upon the length of the saturated acyl chain and upon spreading temperature. Spreading of SMs with very long saturated acyl chains at temperatures 30-35 degrees below T(m) resulted in condensed films with lower in-plane compressibilities, but consistently larger cross-sectional molecular areas than the condensed phases achieved by spreading at temperatures only 10-20 degrees below T(m). This behavior is discussed in terms of the enhancement of SM lateral aggregation by temperature reduction, a common approach used during domain isolation from biomembranes.

Structural basis for glycosphingolipid transfer specificity.

Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes. The conserved and ubiquitous mammalian glycolipid transfer proteins (GLTPs) serve as potential regulators of cell processes mediated by glycosphingolipids, ranging from differentiation and proliferation to invasive adhesion, neurodegeneration and apoptosis. Here we report crystal structures of apo-GLTP (1.65 Å resolution) and lactosylceramide-bound (1.95 Å) GLTP, in which the bound glycosphingolipid is sandwiched, after adaptive recognition, within a previously unknown two-layer all-α-helical topology. Glycosphingolipid binding specificity is achieved through recognition and anchoring of the sugar-amide headgroup to the GLTP recognition centre by hydrogen bond networks and hydrophobic contacts, and encapsulation of both lipid chains, in a precisely oriented manner within a ‘moulded-to-fit’ hydrophobic tunnel. A cleft-like conformational gating mechanism, involving two interhelical loops and one α-helix of GLTP, could enable the glycolipid chains to enter and leave the tunnel in the membrane-associated state. Mutation and functional analyses of residues in the glycolipid recognition centre and within the hydrophobic tunnel support a framework for understanding how GLTPs acquire and release glycosphingolipids during lipid intermembrane transfer and presentation processes.

Interfacial Interactions of Ceramide with Dimyristoylphosphatidylcholine: Impact of the N-Acyl Chain

The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties.

Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated ‘sandwich’ topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

Organization of ganglioside GM1 in phosphatidylcholine bilayers

Molecules of the ganglioside GM1 are randomly distributed in liquid-crystalline 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. This conclusion is based on a freeze-etch electron microscopic study using ferritin-conjugated cholera toxin and cholera toxin alone as ganglioside labels. The average number of GM1 molecules under a label is calculated by a novel method from the dependence of the fraction of bilayer area covered by the label on the mole fraction of GM1 in the bilayer.

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) at the end of C3-, C5-, C7-, or C9-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me4-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me4-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me4-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and ∼506–515 nm) but also showed the absence of the 620–630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me4-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.