Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions

Chemical tools for modulating autophagy

Autophagy (or self-eating) is a cellular pathway that regulates the lysosomal degradation and recycling of obsolete organelles, long-lived proteins, protein aggregates and pathogens. This process occurs under basal conditions and has a crucial role in cellular development, differentiation, survival and homeostasis. In this review, an overview will be given on the autophagy modulators described up to now with the main purpose of giving the chemists interested in this research field a broad overview on the existing autophagy modulators and the methods employed for their identification. Most of the methods employed to monitor autophagy rely on the detection of the lipidated autophagy marker LC3-II (MAP1LC3, microtubule-associated proteins 1A/1B light chain 3, see below) employing antibodies recognizing both the non-lipidated LC3-I as well as LC3-II.Peer reviewe

Inhibidores de la dihidroceramida desaturasa

Referencia OEPM: P200003045.-- Fecha de solicitud: 19/12/2000.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Inhibidores de la dihidroceramida desaturasa (ver figura en archivo de texto adjunto). Derivados de ciclopropenilceramida como inhibidores de desaturasas caracterizados por la fórmula general (I), en la que R1 puede ser un grupo alquilo, alquenilo, alquinilo, arilo o cualquier heterociclo, n puede tener cualquier valor y estar ramificada o no y contener insaturaciones y R2 y R3 pueden tener el mismo o distinto valor, representan grupos arilo, heteroarilo, alquilo o acilo con una o varias insaturaciones en la cadena, que puede estar ramificada o no, y sustituida con grupos OH. Miembros de esta nueva clase de compuestos han mostrado una extraordinaria actividad como inhibidores de la dihidroceramida desaturasa y de la biosíntesis de ceramida y son de utilidad para el tratamiento de patologías asociadas a aumentos en los niveles de ceramida intracelular.Peer reviewe

Chemical biology: Chemistry as an essential tool in biology

La biologia química, entesa com l’ús d’eines químiques per a l’estudi de fenòmens biològics, ha esdevingut una disciplina fonamental per a investigar les funcions de les proteïnes, el seu paper dins les complexes xarxes de senyalització cel·lular, per a la identificació de noves dianes terapèutiques i, en definitiva, per a estudiar les bases moleculars que regulen els processos biològics, tant en condicions normals com patològiques. En aquest article es detallen mitjançant alguns exemples les característiques d’aquesta àrea de recerca multidisciplinària, que comprèn la química orgànica, la biologia molecular, cel·lular iestructural o la biofísica, i se centra, entre d’altres, en el disseny de molècules amb capacitat per a modular l’activitat i les interaccions de les proteïnes, la síntesi de sondes fluorescents o el desenvolupament de mètodes químics per a la síntesi, modificació i immobilització de proteïnes.Paraules clau: Química biològica, reaccions bioortogonals, síntesi i modificació de proteïnes, proteïnes Ras.Chemical biology relies on the use of chemical tools for the characterization of biological processes. In the last decade, this multidisciplinary research area involving organic chemistry as well as molecular, cellular and structural biology and biophysics, among other fields, has played a key role in the identification of protein function and its importance in complex cellular signalling networks and especially in the elucidation of the molecular principles regulating biological processes in both health and disease. This paper presents some examples and describes some of the major research areas in chemical biology, including the design and synthesis of molecules to modulate protein activity and interactions, the synthesis of fluorescent reporters and the development of chemical methods enabling protein synthesis, modification and immobilization.Keywords: Chemical biology, bioorthogonal reactions, protein synthesis and modification, Ras proteins

Analogs of the dihydroceramide desaturase inhibitor GT11 modified at the amide function: synthesis and biological activities

6 pages, 3 figures, 2 schemes, 1 table.-- PMID: 16211106 [PubMed].-- Printed version published Sep 2005.Dihydroceramide desaturase is the last enzyme in the biosynthesis of ceramide de novo. The cyclopropene-containing sphingolipid GT11 is a competitive inhibitor of dihydroceramide desaturase. The biological effects of chemical modification of the GT11 amide linkage are reported in this article. Either N-methyl substitution or replacement of the amide -carbonyl methylene by oxygen result in inactive compounds. In contrast, both urea (3) and thiourea (4) analogs of GT11, as well as three -ketoamides (5–7), did inhibit the desaturation of N-octanoylsphinganine to N-octanoylsphingosine, although with significantly lower potency than GT11. Furthermore, the -ketoamides 5–7 inhibit the acidic ceramidase with similar potencies (IC50 52–83 µM). Inhibition of the neutral/alkaline ceramidase by these compounds requires around 20-fold higher concentrations. Structure–activity relationships and the biological interest of these compounds are discussed.This work was supported in part by MCYT (grants BQU2002-03737). GT thanks Genzyme for partial support of a CSIC I3P contract and CB thanks MECD for a predoctoral FPU fellowshipPeer reviewe

Procedimiento para la obtención de compuestos derivados de ciclopropenilesfingosina

Referencia OEPM: P200402456.-- Fecha de solicitud: 15/10/2004.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Procedimiento para la obtención de compuestos derivados de ciclopropenilesfingosina. En la presente invención se describe un nuevo procedimiento de síntesis de derivados de ciclopropenilesfingosina, los cuales pueden ser utilizados en la elaboración de composiciones farmacéuticas con distintas aplicaciones.Peer reviewe

Utilización de derivados de ciclopropenilesfingosina para la elaboración de una composición farmacéutica moduladora de la actividad de ceramidasa, y sus aplicaciones

Referencia OEPM: P200302767.-- Fecha de solicitud: 25/11/2003.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Utilización de derivados de ciclopropenilesfingosina para la elaboración de una composición farmacéutica moduladora de la actividad de ceramidasa, y sus aplicaciones. La presente invención describe la actividad moduladora de los derivados de ciclopropenilesfingosina sobre las enzimas ceramidasas. Así, un objeto de la invención es el uso de dichos compuestos en la elaboración de una composición farmacéutica útil para el tratamiento de distintas enfermedades (enf. de Faber, enfermedades cardiovasculares, inflamatorias y cáncer). Además, forma parte de la invención el uso de dichas composiciones farmacéuticas en el tratamiento de las mencionadas enfermedades.Peer reviewe

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.Estudi realitzat a partir d’una estada al Max Planck Institut of Molecular Phisiology, Alemanya, entre 2006 i 2008. S’han realitzat dos projectes. En primer lloc la identificació de nous lligands per a proteïnes gràcies a una nova aproximació que combina l'ús de mètodes computacionals amb el desenvolupament de llibreries basades en productes naturals. Segons aquesta aproximació, la similitud estructural existent entre diferents proteïnes pot ser utilitzada com a punt de partida per l'obtenció de nous lligands. Així, un lligand conegut d'una proteïna pot servir com a punt de partida per a l'obtenció de lligands per altres proteïnes que formin part del mateix "cluster". Aquest projecte que requereix l'ús de mètodes multidisciplinaris com la síntesi orgànica, el desenvolupament d'assaigs in vitro i l'expressió i purificació de proteïnes entre d' altres, ha permès trobar nous inhibidors per a l'enzim DD-ligasa en base a la seva similitud estructural amb diferents kinases com p56lck o cdc2. El segon projecte dur a terme durant aquesta estada postdoctoral s´ha basat en el desenvolupament de nous mètodes de síntesi de proteines lipidades per a utilitzar-les posteriorment com a eina química en estudis bioquímics, biofísics o de l'àmbit de la biologia cel•lular. Concretament el projecte s' ha centrat en el desenvolupament de nous mètodes de síntesi de la proteïna Ras lipidada així com el posterior estudi de la distribució de Ras en els diferents microdominis de membranes que componen les cèl•lules, mitjançant espectroscòpia de fluorescència o microscopi de forca atòmica, entre d'altres