Search

Interferon stimulated gene 15 (ISG15) is an ubiquitin-like protein whose expression and conjugation to targets (ISGylation) is induced by infection, interferon (IFN)-α and -β, ischemia, DNA damage and aging. Attention has historically focused on the antiviral effects of ISGylation, which blocks the entry, replication or release of different intracellular pathogens. However, recently, new functions of ISGylation have emerged that implicate it in multiple cellular processes, such as DNA repair, autophagy, protein translation and exosome secretion. In this Review, we discuss the induction and conjugation of ISG15, as well as the functions of ISGylation in the prevention of infections and in cancer progression. We also offer a novel perspective with regard to the latest findings on this pathway, with special attention to the role of ISGylation in the inhibition of exosome secretion, which is mediated by fusion of multivesicular bodies with lysosomes. Finally, we propose that under conditions of stress or infection, ISGylation acts as a defense mechanism to inhibit normal protein translation by modifying protein kinase R (PKR, also known as EIF2AK2), while any newly synthesized proteins are being tagged and thus marked as potentially dangerous. Then, the endosomal system is re-directed towards protein degradation at the lysosome, to effectively 'lock' the cell gates and thus prevent the spread of pathogens, prions and deleterious aggregates through exosomes.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 20-01-2017, La activación de las células T depende de la capacidad del receptor de células T (TCR) para reconocer antígenos específicos en el contacto del complejo mayor de histocompatibilidad (MHC) de las células presentadoras de antígeno (APC). La unión del TCR al MHC promueve la formación de la sinapsis inmunológica (IS). En este proceso, el TCR y las moléculas que se asocian a él se localizan en el área central de la región de contacto entre la célula T y la APC, en lo que se conoce como el complejo de activación supramolecular central (cSMAC). Las moléculas de adhesión se trasladan al complejo de activación supramolecular periférico (pSMAC). Algunas de las proteínas esenciales en este proceso son los miembros de la familia de proteínas Src (Lck y Fyn). La proteína quinasa Lck fosforila los motivos de activación de inmunorreceptores basados en tirosina del complejo TCR/CD3 permitiendo el reclutamiento de moléculas esenciales para la ruta de activación del TCR y la formación de la IS. La formación de la IS también induce cambios en el citoesqueleto de tubulina, incluyendo la translocación del centrosoma o el centro organizador de microtúbulos (MTOC), hacia la IS, acompañado por el aparato de Golgi, los cuerpos multivesiculares y las mitocondrias. Estos cambios facilitan la secreción polarizada de citoquinas y exosomas hacia la APC. La polarización del MTOC dirige el crecimiento activo de microtúbulos (MT), constituyendo el núcleo de una densa red de MT que regula el tráfico vesicular en la SI. La proteína Aurora A es una serina/treonina quinasa que desempeña un papel crítico en la dinámica del centrosoma y del huso mitótico durante la división celular. Durante la maduración del centrosoma, Aurora A promueve el ensamblaje de MT mediante el reclutamiento de factores nucleadores y estabilizadores de los mismos. Debido a su papel en el control de la dinámica de MT, postulamos que Aurora A podría ejercer un papel importante en la activación de células T durante la formación de la SI. Encontramos que Aurora A se activa tras la estimulación del TCR y se localiza en la IS durante el contacto celular. Por otro lado, tanto la inhibición farmacológica como la depleción génica de Aurora A en células T humanas o de ratón altera severamente la dinámica de MT así como el transporte a la IS de microvesículas que contienen CD3. El bloqueo de Aurora A impide la activación del complejo TCR/CD3, interrumpiendo la activación temprana de las células T así como la expresión de los genes CD69, CD25 y IL-2. La inhibición de Aurora A causa la deslocalización de tirosina quinasa Lck en la región de la SI y disminuye sus niveles de fosforilación, lo que indica que Aurora A es necesaria para mantener a Lck en su forma activa. Estos hallazgos demuestran que Aurora A es una molécula reguladora importante en la señalización temprana y del citoesqueleto de tubulina durante la activación de los linfocitos T., T cell activation depends on the ability of the T cell receptor (TCR) to recognize specific antigens presented in the context of the major histocompatibility complex (MHC) on the antigenpresenting cell (APC). The binding of the TCR to MHC promotes the formation of the immune synapse (IS). In this process, the TCR and its associated molecules localize to a central area of the T-cell-APC contact, the central supramolecular activating complex (cSMAC). Adhesion molecules relocate to the peripheral supramolecular activating complex (pSMAC). Essential proteins in this process are the Src family kinase members (Lck and Fyn). Lck phosphorylates the immunoreceptor tyrosine-based activation (ITAM) motifs of the TCR/CD3 complex leading to the recruitment of key molecules for the downstream signalling pathways and the IS formation. The formation of the IS also triggers changes in the tubulin cytoskeleton, including the translocation of the centrosome or microtubule (MT)-organizing centre (MTOC), to the IS, which is accompanied by the Golgi Apparatus, multivesicular bodies and mitochondria. These changes facilitate the polarized secretion of cytokines and exosomes toward the APC. MTOC polarization orchestrates active MT growth and forms the core of a dense MT network that regulates vesicular traffic at the IS. Aurora A is a serine/threonine kinase that plays a critical role in centrosome and spindle dynamics during mitosis. During centrosome maturation, Aurora A promotes MT assembly by recruiting nucleation and stabilization factors. Due to its role in controlling MT dynamics, we hypothesized that Aurora A may play a role in the activation of T lymphocytes during IS formation. We found that Aurora A is activated upon TCR stimulation and localizes at the IS during contact. Moreover, inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3-bearing vesicles at the IS. Specific targeting of Aurora A impairs activation of the TCR/CD3 complex, preventing early T cell activation and downstream expression of CD69, CD25 and IL-2. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings establish Aurora A as a major regulator of early signaling and the tubulin cytoskeleton during T cell activation.

Different protein kinases control signaling emanating from the T cell receptor (TCR) during antigen-specific T cell activation. Mitotic kinases, e.g. Aurora-A, have been widely studied in the context of mitosis due to their role during microtubule (MT) nucleation, becoming critical regulators of cell cycle progression. We have recently described a specific role for Aurora-A kinase in antigenic T cell activation. Blockade of Aurora-A in T cells severely disrupts the dynamics of MTs and CD3ζ-bearing signaling vesicles during T cell activation. Furthermore, Aurora-A deletion impairs the activation of signaling molecules downstream of the TCR. Targeting Aurora-A disturbs the activation of Lck, which is one of the first signals that drive T cell activation in an antigen-dependent manner. This work describes possible models of regulation of Lck by Aurora-A during T cell activation. We also discuss possible roles for Aurora-A in other systems similar to the IS, and its putative functions in cell polarization.

The immune synapse (IS) is a specialized structure established between different immune cells that fulfills several functions, including a role as a communication bridge. This intimate contact between a T cell and an antigen-presenting cell promotes the proliferation and differentiation of lymphocytes involved in the contact. T-cell activation requires the specific triggering of the T-cell receptor (TCR), which promotes the activation of different signaling pathways inducing the polarization of the T cell. During this process, different adhesion and signaling receptors reorganize at specialized membrane domains, concomitantly to the polarization of the tubulin and actin cytoskeletons, forming stable polarization platforms. The centrosome also moves toward the IS, driving the movement of different organelles, such as the biosynthetic, secretory, degrading machinery, and mitochondria, to sustain T-cell activation. A proper orchestration of all these events is essential for T-cell effector functions and the accomplishment of a complete immune response.