MALDI-MS Patterning of Caspase Activities and Its Application in the Assessment of Drug Resistance.

Mass spectrometry (MS) has been widely used for enzyme activity assays. Herein, we propose a MALDI-MS patterning strategy for the convenient visual presentation of multiple enzyme activities with an easy-to-prepare chip. The array-based caspase-activity patterned chip (Casp-PC) is fabricated by hydrophobically assembling different phospholipid-tagged peptide substrates on a modified ITO slide. The advantages of amphipathic phospholipids lead to high-quality mass spectra for imaging analysis. Upon the respective cleavage of these substrates by different caspases, such as caspase-1, -2, -3, and -8, to produce a mass shift, the enzyme activities can be directly evaluated by MALDI-MS patterning by m/z-dependent imaging of the cleavage products. The ability to identify drug-sensitive/resistant cancer cells and assess the curative effects of anticancer drugs is demonstrated, indicating the applicability of the method and the designed chip.

High resolution in vivo 31P-MRS of the liver: potential advantages in the assessment of non-alcoholic fatty liver disease.

BACKGROUND: Biopsy remains the current gold-standard for assessing non-alcoholic fatty liver disease (NAFLD). To develop a non-invasive means of assessing the disease, 31P magnetic resonance spectroscopy (31P-MRS) has been explored, but the severe spectral overlaps and low signal-to-noise-ratio in 31P-MRS spectra at clinical field strength are clearly limiting factors. PURPOSE: To investigate potential advantages of high resolution in vivo 31P-MRS in assessing NAFLD. MATERIAL AND METHODS: The study was conducted at 9.4T in control and carbon tetrachloride (CCl4)-treated rats. Rats were divided according to histopathologic findings into a control group (n = 15), a non-alcoholic steatohepatitis group (n = 17), and a cirrhosis group (n = 12). Data were presented with different reference peaks that are commonly used for peak normalization such as total phosphorous signal, phosphomonoester + phosphodiester (PME + PDE), and nucleotide triphosphate (NTP). Then, multivariate analyses were performed. RESULTS: In all spectra PME and PDE were well resolved into phosphoethanolamine (PE) and phosphocholine (PC), and into glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC), respectively. Those MRS measures quantifiable only in highly resolved spectra had higher correlations with histology than those conventional MRS measures such as PME, PDE, and NTP. The optimized partial least-squares discriminant analysis (PLS-DA) model correctly classified 79% (22/28) of the rats in the training set and correctly predicted 69% (11/16) of the rats in the test set. CONCLUSION: PE, PC, GPE, GPC, and nicotinamide adenine dinucleotide phosphate (NADP) that can be separately quantifiable in highly resolved spectra may further improve the potential efficacy of 31P-MRS in the diagnosis of NAFLD.

The cost of living in the membrane: a case study of hydrophobic mismatch for the multi-segment protein LeuT.

Many observations of the role of the membrane in the function and organization of transmembrane (TM) proteins have been explained in terms of hydrophobic mismatch between the membrane and the inserted protein. For a quantitative investigation of this mechanism in the lipid-protein interactions of functionally relevant conformations adopted by a multi-TM segment protein, the bacterial leucine transporter (LeuT), we employed a novel method, Continuum-Molecular Dynamics (CTMD), that quantifies the energetics of hydrophobic mismatch by combining the elastic continuum theory of membrane deformations with an atomistic level description of the radially asymmetric membrane-protein interface from MD simulations. LeuT has been serving as a model for structure-function studies of the mammalian neurotransmitter:sodium symporters (NSSs), such as the dopamine and serotonin transporters, which are the subject of intense research in the field of neurotransmission. The membrane models in which LeuT was embedded for these studies were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid, or 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids. The results show that deformation of the host membrane alone is not sufficient to alleviate the hydrophobic mismatch at specific residues of LeuT. The calculations reveal significant membrane thinning and water penetration due to the specific local polar environment produced by the charged K288 of TM7 in LeuT, that is membrane-facing deep inside the hydrophobic milieu of the membrane. This significant perturbation is shown to result in unfavorable polar-hydrophobic interactions at neighboring hydrophobic residues in TM1a and TM7. We show that all the effects attributed to the K288 residue (membrane thinning, water penetration, and the unfavorable polar-hydrophobic interactions at TM1a and TM7), are abolished in calculations with the K288A mutant. The involvement of hydrophobic mismatch is somewhat different in the functionally distinct conformations (outward-open, occluded, inward-open) of LeuT, and the differences are shown to connect to structural elements (e.g., TM1a) known to play key roles in transport. This finding suggests a mechanistic hypothesis for the enhanced transport activity observed for the K288A mutant, suggesting that the unfavorable hydrophobic-hydrophilic interactions hinder the motion of TM1a in the functionally relevant conformational transition to the inward-open state. Various extents of such unfavorable interactions, involving exposure to the lipid environment of adjacent hydrophobic and polar residues, are common in multi-segment transmembrane proteins, and must be considered to affect functionally relevant conformational transitions.

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts.

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.

Quantitative experimental assessment of macromolecular crowding effects at membrane surfaces.

We examined how crowding of the surfaces of lipid vesicles with either grafted polyethyleneglycol (PEG) chains or bilayer-anchored protein molecules affects the binding of soluble proteins to the vesicle surface. Escherichia coli dihydrofolate reductase (DHFR, 18 kDa) or a larger fusion protein, NusA-DHFR (72 kDa), binds reversibly but with high affinity to a methotrexate-modified lipid (MTX-PE) incorporated into large unilamellar vesicles. Incorporation of phosphatidylethanolamine-PEG5000 into the vesicles strongly decreases the affinity of binding of both proteins, to a degree that varies roughly exponentially with the lateral density of the PEG chains. Covalently coupling maltose-binding protein (MBP) to the vesicle surfaces also strongly decreases the affinity of binding of NusDHFR or DHFR, to a degree that likewise varies roughly exponentially with the surface density of anchored MBP. Surface-coupled MBP strongly decreases the rate of binding of NusDHFR to MTX-PE-incorporating vesicles but does not affect the rate of NusDHFR dissociation. The large magnitudes of these effects (easily exceeding an order of magnitude for moderate degrees of surface crowding) support previous theoretical analyses and suggest that surface-crowding effects can markedly influence a variety of important aspects of protein behavior in membranes.

Relationship between in vitro phospholipidosis assay using HepG2 cells and 2-week toxicity studies in rats.

Drug candidates under development by industry frequently show phospholipidosis as a side-effect in pre-clinical toxicity studies. This study sets up a cell-based assay for drug-induced phospholipidosis (PLD) and its performance was evaluated based on the in vivo PLD potential of compounds in 2-week toxicity studies in rats. When HepG2 cells were exposed simultaneously to PLD-inducing chemicals and a phospholipid having a fluorophore, an accumulation of phospholipids was detected as an increasing fluorescent intensity. Amiodarone, amitriptyline, fluoxetine, AY-9944, and perhexiline, which are common PLD-inducing chemicals, increased the fluorescent intensity, but acetaminophen, ampicillin, cimetidine, famotidine, or valproic acid, which are non-PLD-inducing chemicals, did not. The fluorescent intensity showed concordance with the pathological observations of phospholipid lamellar bodies in the cells. Then to confirm the predictive performance of the in vitro PLD assay, the 32 proprietary compounds characterized in 2-week toxicity studies in rats were evaluated with this in vitro assay. Because this in vitro assay was vulnerable to cytotoxicity, the innate PLD potential was calculated for each compound. A statistically significant increase in the in vitro PLD potential was seen for the compounds having in vivo PLD-inducing potential in the rat toxicity studies. The results suggest that the in vitro PLD potential could be appropriate to detect the appearance of PLD as a side effect in pre-clinical toxicity studies in rats.

Analysis of protein binding to receptor-doped lipid monolayers by Monte Carlo simulation.

This paper presents a Monte Carlo simulation (MCS) method for estimating the parameters that characterize ligand-receptor binding directly from experimentally derived binding isotherms. Binding parameters are estimated by incorporating an MCS algorithm for ligand binding to a two-dimensional receptor array into a nonlinear regression program. The MCS method was tested by analyzing experimental isotherms of avidin binding to biotinylated lipid in Langmuir-Blodgett (LB) monolayers. The MCS-derived cooperativity coefficients and intrinsic association constants for avidin-biotin binding to LB films are correlated strongly (R2 > 0.93) with the binding parameters determined from the same experimental data by a thermodynamic equilibrium binding model (Zhao et al. 1993. Langmuir. 9:3166-3173). This result shows MCS to be an accurate and potentially more versatile method for characterizing biomolecular interactions at surfaces.

Free volume model for lipid lateral diffusion coefficients. Assessment of the temperature dependence in phosphatidylcholine and phosphatidylethanolamine bilayers.

The lipid lateral diffusion coefficients, DT, in fluid-phase phosphatidylcholine and phosphatidylethanolamine bilayers have been analysed in terms of the free-volume diffusion model by fitting the expression: DT = AT exp[- B/(T - T0)] to the observed temperature dependence, where A, B and T0 are the parameters to be optimized. Application of an unconstrained optimization procedure to data obtained from excimer formation (Galla et al. (1979) J. Membrane Biol. 48, 215-236) and from fluorescence photobleaching (Vaz et al. (1985) Biochemistry 24, 781-786) provides statistical evidence for a free-volume model as opposed to a simple Stokes-Einstein model (T0 = 0), only in certain cases. In the instances for which the parameter T0 can be determined with a reasonable degree of accuracy, it is found that this characteristic temperature at which the free volume extrapolates to zero lies below the bilayer gel-to-fluid phase transition temperature and does not coincide with the pre-transition temperature for phosphatidylcholines.