Identificación de moduladores del apoptosoma mediante química combinatoria.

RESUMEN La apoptosis es un proceso importante en una amplia variedad sistemas biológicos, incluyendo el recambio celular normal, el sistema inmunológico y el desarrollo embrionario. La apoptosis inadecuada está implicada en muchas enfermedades incluyendo enfermedades neurodegenerativas tales como las enfermedades de Alzheimer y Huntington, isquemia, desórdenes autoinmunes y varias formas de cáncer. La familia de proteínas Bcl-2 abarca una clase de estructuras homólogas que sirven para inhibir o para activar la apoptosis en un proceso intrincado y a su vez, bien orquestado. Los estímulos apoptósicos inducen la translocación de miembros pro-apoptósicos de la familia de Bcl-2 a la membrana mitochondrial externa donde forman canales iónicos que pueden contribuir a disipar el potencial transmembrana de la mitocondria y favorecer la liberación de citocromo c. En el citosol, esta proteína se une al factor activador de apoptosis (Apaf-1) para formar el complejo denominado apoptosoma que activa una familia de proteasas denominada caspasas. Las caspasas hidrolizan una serie de proteínas clave para la supervivencia celular y la célula muere de forma no necrótica. Estas moléculas moduladoras podrían ser consideradas como agentes "cabeza de serie" para el desarrollo de compuestos que puedan inhibir el crecimiento de células tumorales y/o paliar o aminorar los daños celulares asociados a las enfermedades neurodegenerativas En la tesis doctoral se utiliza la Química Combinatoria para la identificación de moléculas de interés biomédico moduladoras del apoptosoma. Para ello, en primer lugar es necesario poner a punto un ensayo de alto rendimiento que permita utilizar la diana terapéutica, el apoptosoma, y que permita el cribado funcional de un elevado número de moléculas con el objetivo de identificar efectores artificiales del sistema Apaf-1-caspasas. El trabajo presentado en esta tesis incorpora como novedad la utilización de los componentes básicos recombinantes purificados del apoptosoma y la reconstitución de su actividad in vitro para el cribado. Con este ensayo y formato de cribado, lo que se consigue una mejor definición de la diana molecular de búsqueda de moléculas moduladoras. Por otro lado, la posibilidad de manipulación de los distintos componentes del apoptosoma en las reacciones de reconstitución permite llevar a cabo la caracterización del mecanismo de acción de los compuestos actuvos identificados y su sitio de unión. Finalmente se llevo a cabo unestudio in vivo de los compuestos identificados como moduladores. __________________________________________________________________________________________________Protein-protein interactions represent points of chemical intervention for therapeutic gain in the biological processes associated with disease. Apoptosis is an interesting biological process because its importance in a wide variety of biological systems. Inappropriate apoptosis is involved in many human pathologies, including neurodegenerative diseases such as Alzheimer's and Huntington's, ischaemia, autoimmune disorders and several forms of cancer. Diverse apoptotic stimuli, including activation of cell surface death receptors, anticancer agents, irradiation, lack of survival factors, and ischemia induce signaling cascades that all activate a family of cysteine aspartyl proteases called caspases. It is these proteases that execute the apoptotic process. Effector caspases are responsible for the disassembly of cellular components while initiator caspases are responsible for activation of the effector caspases. Because of the critical consequences of apoptosis malfunctioning, the activation of caspases is scrupulously controlled. Some apoptotic signals activate the mitochondria-mediated or intrinsic pathway that utilizes caspase-9 as its initiator. Caspase-9 activation is triggered by the release to the cytosol of proapoptotic proteins from the mitochondrial inter-membrane space, in particular cytochrome c. The formation of the macromolecular complex named apoptosome is a key event in this pathway. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9. In this macromolecular complex apoptosome-associated caspase-9 is activated and then, in turn, activate effector caspases. To identify molecules that could ameliorate disease-associated apoptosis, drug discovery efforts have initially targeted caspase activity rather than activation. Nevertheless, protein-protein interactions upstream of caspase activation can be also relevant points of intervention for the development of modulators of apoptosis pathways. In particular, recent data propose the formation of the apoptosome as an interesting target for the development of apoptotic modulators. In the absence of detailed structural information, the conventional methods used for the identification of modulators of the apoptosome have been based in indirect measurements of the cytochrome c- and dATP-induced activation of caspase-3-like activity on defined cytosolic extracts. Using this methodology Lademann et al. have identified inhibitors of the apoptosome through the screening of small molecules using cytosolic extracts of selected cells. We have carried out a discovery program employing an in vitro reconstituted active apoptosome assembled from its recombinant constituent proteins. Here we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library of N-alkylglycines. The active compounds rescued from the library were chemically optimized to obtain molecules that bind to both recombinant and human endogenous Apaf-1 and decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis

Frustration induced phases in migrating cell clusters

Collective motion of cells is common in many physiological processes, including tissue development, repair, and tumor formation. Recent experiments have shown that certain malignant cancer cells form clusters in a chemoattractant gradient, which display three different phases of motion: translational, rotational, and random. Intriguingly, all three phases are observed simultaneously, with clusters spontaneously switching between these modes of motion. The origin of this behavior is not understood at present, especially the robust appearance of cluster rotations. Guided by experiments on the motion of two-dimensional clusters in-vitro, we developed an agent based model in which the cells form a cohesive cluster due to attractive and alignment interactions but with potentially different behaviors based on their local environment. We find that when cells at the cluster rim are more motile, all three phases of motion coexist, in excellent agreement with the observations. Using the model we can identify that the transitions between different phases are driven by a competition between an ordered rim and a disordered core accompanied by the creation and annihilation of topological defects in the velocity field. The model makes definite predictions regarding the dependence of the motility phase of the cluster on its size and external chemical gradient, which agree with our experimental data. Our results suggest that heterogeneous behavior of individuals, based on local environment, can lead to novel, experimentally observed phases of collective motion.Comment: 14 pages, 5 figure

A RAB35-p85/PI3K axis controls oscillatory apical protrusions required for efficient chemotactic migration

How cells move chemotactically remains a major unmet challenge in cell biology. Emerging evidences indicate that for interpreting noisy, shallow gradients of soluble cues a system must behave as an excitable process. Here, through an RNAi-based, high-content screening approach, we identify RAB35 as necessary for the formation of growth factors (GFs)-induced waves of Circular Dorsal Ruffles (CDRs), apically restricted actin-rich migratory protrusions. RAB35 is sufficient to induce recurrent and polarized CDRs that travel as propagating waves, thus behaving as an excitable system that can be biased to control cell steering. Consistently, RAB35 is essential for promoting directed chemotactic migration and chemoinvasion of various cells in response to gradients of motogenic GFs. Molecularly, RAB35 does so by directly regulating the activity of p85/PI3K polarity axis. We propose that RAB35 is a molecular determinant for the control of an excitable, oscillatory system that acts as steering wheel for GF-mediated chemotaxis and chemoinvasion

Wiskott-Aldrich syndrome protein controls antigen-presenting cell-driven CD4+ T-cell motility by regulating adhesion to intercellular adhesion molecule-1

International audienceT-cell scanning for antigen-presenting cells (APC) is a finely tuned process. Whereas non-cognate APC trigger T-cell motility via chemokines and intercellular adhesion molecule-1 (ICAM-1), cognate APC deliver a stop signal resulting from antigen recognition. We tested in vitro the contribution of the actin cytoskeleton regulator Wiskott-Aldrich syndrome protein (WASP) to the scanning activity of primary human CD4(+) T cells. WASP knock-down resulted in increased T-cell motility upon encounter with non-cognate dendritic cells or B cells and reduced capacity to stop following antigen recognition. The high motility of WASP-deficient T cells was accompanied by a diminished ability to round up and to stabilize pauses. WASP-deficient T cells migrated in a normal proportion towards CXCL12, CCL19 and CCL21, but displayed an increased adhesion and elongation on ICAM-1. The elongated morphology of WASP-deficient T cells was related to a reduced confinement of high-affinity lymphocyte function-associated antigen 1 to the mid-cell zone. Our data therefore indicate that WASP controls CD4(+) T-cell motility upon APC encounter by regulating lymphocyte function-associated antigen 1 spatial distribution

Collective cell motility promotes chemotactic prowess and resistance to chemorepulsion

Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion [1, 2]. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination [1]. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates [3]. Whether malignant lymphocytes are capable of coordinating their motility in the context of chemokine gradients is, however, unknown. Here, we show that, upon exposure to CCL19 or CXCL12 gradients, malignant B and T lymphocytes assemble into clusters that migrate directionally and display a wider chemotactic sensitivity than individual cells. Physical modeling recapitulates cluster motility statistics and shows that intracluster cell cohesion results in noise reduction and enhanced directionality. Quantitative image analysis reveals that cluster migration runs are periodically interrupted by transitory rotation and random phases that favor leader cell turnover. Additionally, internalization of CCR7 in leader cells is accompanied by protrusion retraction, loss of polarity, and the ensuing replacement by new leader cells. These mechanisms ensure sustained forward migration and resistance to chemorepulsion, a behavior of individual cells exposed to steep CCL19 gradients that depends on CCR7 endocytosis. Thus, coordinated cluster dynamics confer distinct chemotactic properties, highlighting unexpected features of lymphoid cell migration

Compuestos para la inhibición de la apoptosis

La presente invención se refiere a compuestos que actúan como inhibidores de la apoptosis, así como a procedimientos para su preparación, a composiciones farmacéuticas que los contienen y a su uso en medicina.Peer reviewedLaboratorios SALVAT SA, Consejo Superior de Investigaciones Científicas (España)T3 Traducción de patente europe

CIP4 controls CCL19-driven cell steering and chemotaxis in chronic lymphocytic leukemia

Solid tumor dissemination relies on the reprogramming of molecular pathways controlling chemotaxis. Whether the motility of non-solid tumors such as leukemia depends on the deregulated expression of molecules decoding chemotactic signals remains an open question. We identify here the membrane remodeling F-BAR adapter protein CIP4 as a key regulator of chemotaxis in chronic lymphocytic leukemia (CLL). CIP4 is expressed at abnormally high levels in CLL cells where it is required for CCL19-induced chemotaxis. Upon CCL19 stimulation of CLL cells, CIP4 associates with GTP-bound Cdc42 and is recruited to the rear of the lamellipodium and along microspikes radiating through the lamellipodium. Consistent with its cellular distribution, CIP4 removal impairs both the assembly of the polarized lamellipodium and directional migration along a diffusible CCL19 gradient. Furthermore, CIP4 depletion results in decreased activation of WASP, but increased activation of PAK1 and p38 MAPK. Notably, p38 MAPK inhibition results in impaired lamellipodium assembly and loss of directional migration. This suggests that CIP4 modulates both the WASP and p38 MAPK pathways to promote lamellipodium assembly and chemotaxis. Overall, our study reveals a critical role of CIP4 in mediating chemotaxis of CLL cells, by controlling the dynamics of microspike-containing protrusions and cell steering

Small molecule inhibitors of Apaf-1-related caspase-3/-9 activation that control mitochondrial-dependent apoptosis

10 pages, 5 figures.-- PMID: 16341125 [PubMed].-- Available online Dec 9, 2005.Supporting information available at: http://www.nature.com/cdd/journal/v13/n9/suppinfo/4401828s1.html?url=/cdd/journal/v13/n9/abs/4401828a.htmlApoptosis is a biological process relevant to human disease states that is strongly regulated through protein–protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis.This work was supported by grants from Spanish Ministry of Science and Techonology (SAF2001-2811, SAF2001-2286 and BIO2004-998), Fundación Areces and Fundación Valenciana de Investigaciones Biomédicas.Peer reviewe