Many translated example sentences containing “centro organizador” – English- Spanish centrosoma es el único centro organizador de microtubulos [ ]. tema el citoesqueleto. ‘red de filamentos proteicos con función esquelética que constituyen el “andamio” interno de la célula’. se encuentra en todas las. los microtúbulos están anclados por sus extremos negativos al centro organizador de microtúbulos y los extremos positivos se dirigen hacia la periferia ; durante.
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Method of identifying compounds regulators microubulos deacetylase activity of HDAC6, and applications. The object of the present invention is a method of identification of regulatory compounds of the tubulin deacetylase activity of HDAC6 protein, and use of such regulatory compounds in preparing medicaments or pharmaceutical compositions for treating diseases associated with altered activation of T lymphocytes.
The development of these methods is based on the regulation of the levels of tubulin cento using HDAC6 tubulin deacetylase as a therapeutic target.
Therefore, it is ascribed to the pharmacological and biomedical sector for application in the health sector. And, more specifically to the field of methods of identifying novel therapeutic compounds and pharmaceutical compositions for treating diseases or disorders splenic immune system disorders. Therefore, regulation of the immune response is crucial in the treatment of the most important causes of morbidity and mortality in our society.
Activation of T cells requires antigen binding properly presented by molecules of the major histocompatibility complex antigen presenting cells. Three-dimensional seggregation of supramolecular activation clusters in T cells. Nature; Grakoui, A.
Three-dimensional supramolecular activation clusters of seggregation in T cells. Nature82 ; Grakoui, Cenfro. Nature Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells. The area of attachment of cytotoxic T lymphocytes to their target cells shows high motility and polarization of actin, but not myosin. The specific direct interaction of helper T cells and antigen-presenting B cells.
Activation of T cells by antigen also involves translocation microtubule organizing center MTOC and the Golgi apparatus to the site of contact with antigen presenting cell and the production of IL-2 Geiger, B. Cell Biol 95, ; Exclusion of CD43 from the immunological synapse is mediated by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin.
Immunity 15; Roumier, A. Allenspach, EJ, Cullinan, P. The membrane-microfilament linker ezrin is involved in the formation of the immunological synapse and in T cell activation. The membrane-microfilament linker ezrin is Involved in the formation of the immunological synapse and in T cell activation. Immunity 15 Structural insights into microtubule function. However, to date, little was known about the role of microtubules in the development of the immune response Nogales, E. Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.
Biochemistry fe; Piperno, G.
Heterogeneity Among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin J. Cell Biol, 98, ; HDAC6 is a microtubule-associated deacetylase. Nature; Matsuyama, A. In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. In vivo destabilization of microtubules by dynamic HDAC6-mediated deacetylation. Three proteins define a microtubupos of human histone-deacetylases related to yeast Hdalp; Proc. Domain selective small-molecule inhibitor of histone deacetylase 6 HDAC6 -mediated tubulin deacetylation.
USA Three proteins define a class of human histone deacetylases-Hdalp related to yeast; This enzyme deacetylated tubulin in cells and its activity it can be inhibited by trichostatin A and tubacin pharmacological agents Hubbert, C. Furthermore, we should highlight the wide range of cellular activities that HDACs are involved that are appearing in the prior art and have enabled the establishment of new therapeutic approaches to many human pathologies, by using inhibitors and agonists HDACs, and the development of new methods of identification and evaluation of new therapeutic compounds.
Thus, the following applications have been described as therapeutic target protein crntro from the functional activities involved in: Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy, J. Cytoskeletal rearrangement during migration and activation of T lymphocytes.
The immunological synapse and the actin cytoskeleton: Actin microfilaments are considered the most important components organizsdor activation of T cells Valitutti, SM, Dessing, M.
Citoesqueleto – Wikipedia, a enciclopedia libre
Exp Med; Sawing, JM, Nieto, Organizxdor. However, there are increasing evidence that other cytoskeletal proteins involved in this process, but to date has not linked the microtubule acetylation with immune pathology. Therefore, the regulation of desacetiladora activity tubulin mediated HDAC6 and its implications for modulating the activation of T cells, disclosed herein for the first time, it can be an effective treatment for diseases associated with an immune response and also exacerbated in those that occur with a deficient immune response.
The object of the present invention is a method of identifying and evaluating the activity regulating compounds HDAC6 protein on T cell activation, comprising the following steps: The results of the present invention demonstrate that microtubules also play a role in early and late activation of T cell receptor antigen binding T cell modifies microtubule acetylation in a dynamic way initially They are deacetylated, for about 15 minutes after gradually reacetilen.
In addition, the uncontrolled activation of HDAC6 mifrotubulos translocation of secretory apparatus to the antigen presenting cell and decreases the production of IL-2, which are two events of early and late, respectively activation.
In summary, the present invention is based on the discovery that mifrotubulos acetylation is important to modulate the activation of Mlcrotubulos cells and is regulated by the HDAC6 protein, allowing define new therapeutic approaches for treating diseases splenic immune system disorders.
These therapeutic approaches are fentro on the use of compounds or buffering agents, inhibitors and activators, the activity of said protein HDAC6. Compounds inhibitors or antagonists of HDAC6, which consequently produce a net tubulin acetylation, are useful in restoring impaired immune response.
Moreover, activators or agonists HDAC6 compounds could play a role in organozador damage associated with an exacerbated immune responses such as those taking place in an autoimmune disease.
In microtubulis regard, the gene transfer technology could, by viral vectors, gene be useful in therapy to express tissue-specific manner HDAC6 enzyme in T lymphocytes of patients suffering from such diseases. Thus, an object of the present centrp is the use of a compound or regulating agent, inhibitor or activator of the activity of HDAC6 protein, hereinafter use of a compound of the present invention, in preparing medicaments or pharmaceutical compositions for the treatment of diseases, disorders or diseases of mammals, preferably human, that develop with alterations of the immune system, preferably with the activation of T cells altered.
This definition also includes those compounds that prevent or decrease expression of the gene encoding the HDAC6 protein, ie, preventing or diminuyen gene transcription, the maturation of mRNA, mRNA translation orgaanizador post-translational modification.
This definition also includes those compounds or molecules that increase expression of the gene encoding the protein HDAC6.
In the prior art there are described compounds agonists activity of HDAC proteins, which organjzador be used within the scope of this invention as agonists HADC6 protein, and below by way of illustration without this implying a limitation to the scope of the present invention: A particular embodiment of the present invention is the use organizaror an agonist compound in the manufacture of a medicament or therapeutic composition for treating a disease associated with an exacerbation of the immune organkzador based on the compound it consists of a genetic construct allowing expression of the human HDAC6 SEQ ID NO.
Furthermore, the source of these regulators compounds, inhibitors or agonists of the activity of proteins HDACs can be varied, so that can be natural e. An object of the present invention is a method of identifying and evaluating the activity regulating compounds HDAC6 protein on T cell activation, hereinafter method of identifying compounds of the present invention, comprising the following steps: A particular object of the present invention is a method for identifying potential inhibitors of the invention where the point T i lymphocytes can be stimulated or prepared by methods known in the prior art compounds see Example ; stimulation antigen presenting cells Raji cells more SEE and transformation of cell cultures of T cells with a genetic construct allows expression of HDAC6 gene allow better assessment of the effect of such potential inhibitor compounds.
A particular object of the present invention is the method of identifying compounds of the invention in which the determination of the levels of tubulin acetylation ii can be performed by techniques immunoblot or orgahizador blot with appropriate antibodies see Example 2 and Figure 2.
LeDizet M, Piperno G. M LeDizet Piperno G. These procedures can be applied for the identification of regulatory compounds, inhibitors and agonists, HDAC6 protein as deacetylase protein, and subsequent measurement of buffer capacity of such compounds on T lymphocyte activation.
Another particular object of the present invention is the method of identifying compounds of the invention in which the determination of a parameter related to the activation of T lymphocytes can be performed, for illustrative purposes and without limiting the scope of the present invention, by a technique belonging to the following group: Furthermore, the origin of these compounds candidates for the identification method of the invention can be varied so that can be natural e.
Centro organizador dos microtúbulos
Also, from the results of the present invention the importance of acetyl-transferases proteins, which act by increasing levels of tubulin acetylation observed during activation of T cells, and allow it is found: Figura 1 Figure 1. Raji cells were contacted with J77 cells on fibronectin-coated coverslips.
Then the cells were fixed, permeabilized and stained with antibodies as indicated. Ghosting and phase contrast are displayed on the right. Asterisks indicate the location of Raji cells in cell conjugates. Figura 2 Figure 2. Then they detected by immunoblot levels acetylated tubulin, tubulin and vimentin in cell lysates under non-reducing conditions as described in Methods. Then levels of tubulin and acetylated tubulin were detected by immunoblotting under non-reducing conditions.
J77 and Raji cells were contacted in the presence of SEE or SEB for the indicated times, and then the levels of acetylated tubulin and tubulin by immunoblot under nonreducing conditions were detected. Figura 3 Figure 3. Then the cells were fixed, permeabilized and stained for HDAC6 and acetylated tubulin. The arrows indicate the microtubule organizing centers. Acetylated microtubules were stained in conjugates between T cells and antigen presenting cells.
Los marcadores de peso molecular se indican a la izquierda. The molecular weight markers are indicated on the left.
CENTRO ORGANIZADOR DE LOS MICROTÚBULOS by Sara De La Peña on Prezi
Se muestra un experimento representativo de tres. A representative experiment of three is shown.
Then the cell conjugates fixed, permeabilized and stained for acetylated tubulin. Arrowheads are directed towards the point of contact of the T cells with antigen presenting cells. Figura 4 Figure 4. Raji cells were labeled with blue probe CMAC. El tiempo transcurrido desde el contacto inicial en minutos se indica en la esquina superior izquierda de cada imagen.
The time elapsed since the initial contact time is indicated in minutes in the upper left corner of each image. Asterisks denote Raji cells. Figura 5 Figure 5. Asterisks indicate the location of the antigen presenting cells in the cell conjugates. Figura 6 Figure 6. Then the cells were stained for LFA-1 and analyzed by confocal microscopy.