A. Ulises Acuña

The Conformation of Serum Albumin in Solution: A Combined Phosphorescence Depolarization-Hydrodynamic Modeling Study

There is a striking disparity between the heart-shaped structure of human serum albumin (HSA) observed in single crystals and the elongated ellipsoid model used for decades to interpret the protein solution hydrodynamics at neutral pH. These two contrasting views could be reconciled if the protein were flexible enough to change its conformation in solution from that found in the crystal. To investigate this possibility we recorded the rotational motions in real time of an erythrosin-bovine serum albumin complex (Er-BSA) over an extended time range, using phosphorescence depolarization techniques. These measurements are consistent with the absence of independent motions of large protein segments in solution, in the time range from nanoseconds to fractions of milliseconds, and give a single rotational correlation time phi(BSA, 1 cP, 20 degrees C) = 40 +/- 2 ns. In addition, we report a detailed analysis of the protein hydrodynamics based on two bead-modeling methods. In the first, BSA was modeled as a triangular prismatic shell with optimized dimensions of 84 x 84 x 84 x 31.5 A, whereas in the second, the atomic-level structure of HSA obtained from crystallographic data was used to build a much more refined rough-shell model. In both cases, the predicted and experimental rotational diffusion rate and other hydrodynamic parameters were in good agreement. Therefore, the overall conformation in neutral solution of BSA, as of HSA, should be rigid, in the sense indicated above, and very similar to the heart-shaped structure observed in HSA crystals.

Intracellular Triggering of Fas Aggregation and Recruitment of Apoptotic Molecules into Fas-enriched Rafts in Selective Tumor Cell Apoptosis

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.

Liquid-crystalline phases of cholesterol/lipid bilayers as revealed by the fluorescence of trans-parinaric acid.

The presence of two liquid-crystalline phases, alpha and beta, in mixed bilayers of dimyristoylphosphatidylcholine/cholesterol was detected by the changes in the distribution of the fluorescence lifetimes of t-PnA, as analyzed by the Maximum Entropy Method. The formation of the liquid-ordered beta-phase, in the 30-40 degrees C temperature range as a function of cholesterol concentration (0-40 mol%), could be related quantitatively to the relative amplitude of a long lifetime component of the probe (10-14 ns). Based on this evidence, the phase behavior of mixtures of the unsaturated lipid palmitoyloleoylphosphatidylcholine and cholesterol was determined using the same technique, for cholesterol concentrations in the 0-50 mol% range, between 10 and 40 degrees C. It was found that two liquid-crystalline phases are also formed in this system, with physical properties reminiscent of the alpha- and beta-phases formed with saturated lipids. However, in this case it was determined that, for temperatures in the physiological range, the alpha- and beta-phases coexist up to 40 mol% cholesterol. This finding may be of significant biological relevance, because it supports the long held notion that cholesterol is responsible for the lipid packing heterogeneity of several natural membranes rich in unsaturated lipid components.

Protein self-association in crowded protein solutions: a time-resolved fluorescence polarization study.

The self‐association equilibrium of a tracer protein, apomyoglobin (apoMb), in highly concentrated crowded solutions of ribonuclease A (RNase A) and human serum albumin (HSA), has been studied as a model system of protein interactions that occur in crowded macromolecular environments. The rotational diffusion of the tracer protein labeled with two different fluorescent dyes, 8‐anilinonaphthalene‐1‐sulfonate and fluorescein isothiocyanate, was successfully recorded as a function of the two crowder concentrations in the 50–200 mg/mL range, using picosecond‐resolved fluorescence anisotropy methods. It was found that apoMb molecules self‐associate at high RNase A concentration to yield a flexible dimer. The apparent dimerization constant, which increases with RNase A concentration, could also be estimated from the fractional contribution of monomeric and dimeric species to the total fluorescence anisotropy of the samples. In contrast, an equivalent mass concentration of HSA does not result in tracer dimerization. This different effect of RNase A and HSA is much larger than that predicted from simple models based only on the free volume available to apoMb, indicating that additional, nonspecific interactions between tracer and crowder should come into play. The time‐resolved fluorescence polarization methods described here are expected to be of general applicability to the detection and quantification of crowding effects in a variety of macromolecules of biological relevance.

Synthesis of BODIPY-labeled alkylphosphocholines with leishmanicidal activity, as fluorescent analogues of miltefosine

Two general synthetic methods are described, by which the highly fluorescent and photostable BODIPY group can be inserted in and aligned with the alkyl backbone of linear lipids. These methods have been used to prepare strongly emitting analogues of the leishmanicidal drug miltefosine, in which the antiparasite activity in vitro of the original drug is preserved.

Synthesis and biological evaluation of fluorescent leishmanicidal analogues of hexadecylphosphocholine (miltefosine) as probes of antiparasite mechanisms.

The leishmanicidal mechanism of miltefosine (hexadecylphosphocholine, MT) is not clearly understood. Valuable insights into its mode of action could be obtained by fluorescence techniques, given suitably emitting analogues. In this regard, the synthesis and biological characterization of two fully competent MT fluorescent analogues is reported here: all-(E)-13-phenyltrideca-6,8,10,12-tetraenylphosphocholine (PTE-MT) and all-(E)-13-phenyltrideca-8,10,12-trien-6-ynylphosphocholine (PTRI-MT). Both compounds show large absorption coefficients and a modest, but usable, fluorescence yield. Their activities were very similar to that of MT and were recognized by the MT uptake system of Leishmania. Their localization in living L. donovani promastigotes by confocal microscopy show a homogeneous intracellular distribution of the fluorescence. The concentration of PTRI-MT within the parasites (ca. 1.7 mM) showed a 100-fold enrichment relative to its external concentration. These results are consistent with a multiple target leishmanicidal mechanism for MT and validate the application of these analogues for pharmacokinetic and diagnostic studies concerning the chemotherapy of leishmaniasis.

The transverse location of the fluorescent probe trans-parinaric acid in lipid bilayers

The transverse location of trans-parinaric acid in spherical vesicles made up from dipalmitoylphosphatidylcholine has been investigated by the differential quenching of the probe fluorescence by 5- and 16-doxylstearic acid derivatives. The quenching data are interpreted in terms of a local fluorophore concentration factor. In this way it was found that the polyene of t-PnA is located within the inner part of the bilayer (presumably aligned with the bilayer lipids), both in the gel and in the liquid crystalline phases.

Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measurements are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature.

Drug uptake, lipid rafts and vesicle trafficking modulate resistance to an anticancer lysophosphatidylcholine analogue in yeast

Background: The antitumor lipid edelfosine kills yeast by inducing selective internalization of raft-associated proteins. Results: Impairing vesicular trafficking to the vacuole counteracted edelfosine-induced plasma membrane alterations without affecting internalization of the drug. Conclusion: Recycling of raft-associated proteins to the plasma membrane prevents edelfosine cytotoxicity. Significance: Vesicular trafficking is a critical process mediating edelfosine resistance in yeast that could be extrapolated to tumor cells. The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.

New Transmembrane Polyene Bolaamphiphiles as Fluorescent Probes in Lipid Bilayers

Inspired by Archaebacterial lipids, transmembrane probes anchor a sensing fluorescent polyene with Ångström resolution deep within a lipid layer. These new bolaamphiphiles are obtained in good yields from a double cross-coupling between esters with a terminal acetylene group and conjugated 1,ω-dihalopolyenes, followed by partial reduction of the triple bond.