M. Pilar Lillo

Structural basis of the interaction between integrin α6β4 and plectin at the hemidesmosomes

The interaction between the integrin α6β4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 Å resolution of the primary α6β4–plectin complex, formed by the first pair of fibronectin type III domains and the N‐terminal region of the connecting segment of β4 and the actin‐binding domain of plectin. Two missense mutations in β4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 Å resolution of the β4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of β4 on binding to plectin. This conformational switch is correlated with the way α6β4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.

Endocannabinoids and cannabinoid analogues block cardiac hKv1.5 channels in a cannabinoid receptor-independent manner.

AIMS Endocannabinoids are synthesized from lipid precursors at the plasma membranes of virtually all cell types, including cardiac myocytes. Endocannabinoids can modulate neuronal and vascular ion channels through receptor-independent actions; however, their effects on cardiac K(+) channels are unknown. This study was undertaken to determine the receptor-independent effects of endocannabinoids such as anandamide (N-arachidonoylethanolamine, AEA), 2-arachidonoylglycerol (2-AG), and endocannabinoid-related compounds such as N-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA), the endogenous lipid lysophosphatidylinositol (LPI), and the fatty acids from which some of these compounds are endogenously synthesized, on human cardiac Kv1.5 channels, which generate the ultrarapid delayed rectifier current (I(Kur)). METHODS AND RESULTS hKv1.5 currents (I(hKv1.5)) were recorded in mouse fibroblasts (Ltk(-) cells) by using the whole-cell patch-clamp technique. Most of these compounds inhibited I(hKv1.5) in a concentration-dependent manner, the potency being determined by the number of C atoms in the fatty acyl chain. Indeed, AEA and 2-AG, which are arachidonic acid (20:4) derivatives, exhibited the highest potency (IC(50) approximately 0.9-2.5 microM), whereas PEA, a palmitic acid (PA-16:0) derivative, exhibited the lowest potency. The inhibition was independent of cannabinoid receptor engagement and of changes in the order and microviscosity of the membrane. Furthermore, blockade induced by AEA and 2-AG was abolished upon mutation of the R487 residue, which determines the external tetraethylammonium sensitivity and is located in the external entryway of the pore. AEA significantly prolonged the duration of action potentials (APs) recorded in mouse left atria. CONCLUSION These results indicate that endocannabinoids block human cardiac Kv1.5 channels by interacting with an extracellular binding site, a mechanism by which these compounds regulate atrial AP shape.

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.

Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K(+) channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K(+) current (I(to1)). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I(Kv4.3) in a concentration-dependent manner, AEA and 2-AG being the most potent (IC(50) approximately 0.3-0.4 microM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I(to1) recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.

Irvalec inserts into the plasma membrane causing rapid loss of integrity and necrotic cell death in tumor cells.

Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca2+ influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn2+. Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity.

Cholesterol effect on the physical state of lipid multibilayers from the platelet plasma membrane by time-resolved fluorescence

There are indications that the plasma membrane lipid composition and, in particular, the cholesterol/phospholipid (C/PL) ratio, affects platelet function. As a first approximation to the molecular characterization of the effect of cholesterol on the order, fluidity and lateral heterogeneity of the platelet plasma membrane, the steady-state and time-resolved fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trans-parinaric acid (tPnA) has been studied in multibilayer vesicles of phospholipids extracted from human platelet plasma membrane with different cholesterol/phospholipid molar ratios modified in vitro from 0.07 to 0.9. The DPH studies show that the increased presence of cholesterol has a stronger effect on the order than on the fluidity of the bilayer, as has been previously observed in other lipid membranes. On the other hand, from the analysis of the fluorescence kinetics of tPnA we conclude that a higher cholesterol content gives rise to an increase of the heterogeneity of the bilayer, due to a larger fraction of solid-like lipid domains. These domains contain a cholesterol concentration much higher than the macroscopic average value.

Characterization of the Control Catabolite Protein of Gluconeogenic Genes Repressor by Fluorescence Cross-Correlation Spectroscopy and Other Biophysical Approaches

Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a prerequisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control catabolite protein of gluconeogenic genes, a key metabolic regulator in Gram-positive bacteria, with two oligonucleotides derived from its target operator sequences, gapB and pckA. According to our FCCS experiments, the stoichiometry of binding is twofold larger for the pckA target than for gapB. Correcting the FCCS data for protein self-association indicated that control catabolite protein of gluconeogenic genes forms dimeric complexes on the gapB target and tetrameric complexes on the pckA target. Analytical ultracentrifugation coupled with fluorescence anisotropy and hydrodynamic modeling allowed unambiguous confirmation of this result. The use of multiple complementary techniques to characterize these complexes should be employed wherever possible. However, there are cases in which analytical ultracentrifugation is precluded, due to protein stability, solubility, or availability, or, more obviously, when the studies are carried out in live cells. If information concerning the self-association of the protein is available, FCCS can be used for the direct and simultaneous determination of the affinity, cooperativity, and stoichiometry of protein-DNA complexes in a concentration range and conditions relevant to the regulation of these interactions.

Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin

eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a KD of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [14C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin.

Two-Photon Fluorescence Anisotropy Imaging to Elucidate the Dynamics and the Stability of Immobilized Proteins

Time/spatial-resolved fluorescence determines anisotropy values of supported-fluorescent proteins through different immobilization chemistries, evidencing some of the molecular mechanisms that drive the stabilization of proteins at the interfaces with solid surfaces. Fluorescence anisotropy imaging provides a normalized protein mobility parameter that serves as a guide to study the effect of different immobilization parameters (length and flexibility of the spacer arm and multivalency of the protein-support interaction) on the final stability of the supported proteins. Proteins in a more constrained environment correspond to the most thermostable ones, as was shown by thermal inactivation studies. This work contributes to explain the experimental evidence found with conventional methods based on observable measurements; thus this advanced characterization technique provides reliable molecular information about the immobilized proteins with sub-micrometer spatial resolution. Such information has been very useful for fabricating highly stable heterogeneous biocatalysts with high interest in industrial developments.

Elisidepsin Interacts Directly with Glycosylceramides in the Plasma Membrane of Tumor Cells to Induce Necrotic Cell Death.

Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG gene in these deficient cells restored glycosylceramides synthesis, rendering them sensitive to elisidepsin, at a similar level than parental B16 cells. These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death. They also indicate that cell membrane lipids are a plausible target for antineoplastic therapy.