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Identification of the Sumoylation Sites of DaxxHuang, Yi-hsin 03 February 2004 (has links)
SUMO (small ubiquitin-like modifier) protein, also known as Smt3
(suppressor of Mif2 protein 3) of Saccharomyces cerevisiae is an
ubiquitin-like protein due to the similar post-transcriptional modifications
to their substrates. There are three members of SUMO genes (SUMO-1,
SUMO-2 & SUMO-3) in the vertebrate, while only one SUMO gene exists
in the invertebrate. Covalent modification of cellular proteins by the
SUMO regulates various cellular processions, such as nuclear transport,
transcription repression and cellular apoptosis. To investigate the biological
functions of SUMO-1 and SUMO-2, yeast two hybrid assays were applied.
Results showed that N-terminus (Daxx1, 1-282 amino acids) and
C-terminus (Daxx4, 607-740 amino acids) of Daxx were the SUMOs
interacting fragments.
For identification of the sumoylation site on Daxx1 and Daxx4, six
mutants (K60R, K630A, K631A, K634A, K630, 631A and K630, 631,
634A) were constructed. In vitro sumoylation were applied in the Daxx1
fragment and mutated Daxx1 (K60R) as well as the Daxx4 fragment and
mutated Daxx4 (K630A, K631A , K634A, K630, 631A and K630, 631,
634A) to identify the sumoylation sites of Daxx.
Our results showed that Daxx1 K60 was one of the sumoylation sites,
neverthless it was not a major sumoylation site. The major sumoylation
sites were on the C-terminus of Daxx (Daxx4). The major sumoylation sites
of SUMO-1 on Daxx4 seemed different from those of SUMO-2. Mutants
(K631A and K634A) of Daxx4 decreased the yields of sumoylation
complexes of SUMO-1 more than that of Daxx4 K630. However, mutants
(K630A and K631A) of Daxx4 decreased the yields of sumoylation
complexes of SUMO-2 more than that of Daxx4 K634. Thus we propose
that the major sumoylation sites of SUMO-1 on Daxx are K631A and
K634A and that of SUMO-2 are K630A and K631A. Daxx may have other
sumoylation sites on the Daxx C-terminal 635-740 amino acids fragment,
unless the sumoylation reactions of Daxx mutants were pseudo-positive
reactions which might be caused by the improper folding of Daxx4 during
in vitro sumoylation.
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The interactions between Human SMT3 families and Daxx detected by yeast two-hybrid assayChou, Yu-Huai 27 July 2001 (has links)
Abstract
SMT3 (Suppressor of MIF2 3 protein) was identified as a mutation
suppressor in yeast centromere protein MIF2. It is also known as an
ubiquitin-like protein due to the smilarities of their primary structures that
is very conserved during the eukaryotic evolution. Although only one
SMT3 was found in low eukaryotes such as in yeast, three members of
SMT3 (SMT3A, SMT3B and SMT3C) have been identified in high
eukaryotes. It has been known that SMT3C plays an important role in post-translational modification. However, the functions of SMT3A and SMT3B are not well studied yet and the relationship among the SMT3 families remains unclear.
In the present study, Daxx, a Fas binding protein, was demonstrated to bind to SMT3B using yeast two-hybrid assay. It was found that the
N-terminal domain of Daxx (Daxx 1) and the C-terminal domain of Daxx
(Daxx 4), respecifitively, bound to all members of human SMT3
families (including SMT3A, SMT3B and SMT3C). Neverthless,
mechanisms of interactions between the SMT3 families and Daxx
domains remined unclear. Studies on truncated human SMT3 families
have shown that two glycines on the C-terminal end of human SMT3
families were required in the interaction between SMT3 and Daxx
domains, for example, SMT3A and SMT3B required C-terminal two glycines on the Daxx 4 domain where as SMT3C required C-terminal two glycines on the Daxx 1 domain. Morever, truncated SMT3C and Daxx 1 domain point mutations have also indicated that the the linkage of
glycine97 of SMT3C and the lysine60 of Daxx 1, in which the SMT3C/
SUMO-1 consensus sequence £ZKXE was found. Further, SMT3C was
the only member of the SMT3 families capable of self-reacting.
Results also suggested that similar mechanism of interaction
between SMT3A/B and Daxx 1, which is not in accordance with the model proposed in this study regarding the interaction mechanism between SMT3C and Daxx 1. Although two glycines on the C-terminal end of SMT3A/B were necessary for the interactions with Daxx 4 domains, the SMT3C/SUMO-1-consensus sequence £ZKXE was not detected in the Daxx 4 domain. It is therefore, suggested that the mechanism of the interaction between SMT3A/B and Daxx 4 is similar to that of SMT3C and Daxx 1, that may required different binding sequences that is specific for SMT3A/B.
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Développement d'une stratégie thérapeutique anti-apoptotique contre les lésions d'ischémie-reperfusion myocardique / Development of therapeutical anti-apoptotic strategy against myocardial ischemia-reperfusionFranck-Miclo, Alicia 28 September 2012 (has links)
L'infarctus du myocarde (IDM) est consécutif à une occlusion coronaire provoquant une ischémie prolongée. La taille de l'infarctus est le déterminant majeur de la récupération du myocarde et de la survie du patient. La reperfusion coronaire la plus précoce (par thrombolyse ou angioplastie) est reconnue comme étant le seul traitement recommandé. Cependant, malgré les effets bénéfiques, la reperfusion s'accompagne d'effets délétères appelés « lésions de reperfusion ». Récemment, l'efficacité du postconditionnement ischémique (PostC) a été démontrée en clinique. Il permet, lorsqu'il est utilisé au cours de l'angioplastie primaire ou de sauvetage, de réduire significativement la taille de l'infarctus. L'application du protocole de PostC reste cependant limitée aux patients admis dans les centres d'angioplastie, ce qui exclut tous les patients thrombolysés. Afin d'améliorer la cardioprotection, il est nécessaire de déterminer la fenêtre thérapeutique temporelle optimale d'administration du PostC. De plus, il est nécessaire de développer des stratégies thérapeutiques nouvelles, adjuvants de la reperfusion, qui pourront être administrées dès que le diagnostic d'IDM est posé. L'objectif de notre travail a donc été d'étudier l'influence du délai d'application du PostC sur l'efficacité de cardioprotection dans un modèle murin d'ischémie-reperfusion et de rechercher des substances pharmacologiques cardioprotectrices. Nos travaux révèlent que l'efficacité du PostC est maintenue s'il est appliqué les 30 premières minutes après le début de la reperfusion. D'autre part, nous avons évalué le potentiel thérapeutique de peptides inhibiteurs ciblant l'apoptose responsable de la mort cellulaire au cours de l'ischémie-reperfusion. L'utilisation de peptides Tat-BH4 et Pip2b-BH4 pour le traitement des lésions d'ischémie-reperfusion myocardique a fait l'objet d'une étude preuve-de-concept in vivo. Les constructions peptidiques inhibant l'interaction FAS-DAXX, injectées en intraveineuse au moment de la reperfusion, induisent une diminution de 50% de la taille d'infarctus et de l'apoptose. Ils ont fait l'objet d'un dépôt de brevet car ils constituent des outils pharmacologiques à haut potentiel thérapeutique. / Myocardial infarction (MI) results from a coronary occlusion leading to severe ischemia. Infarct size is a major determinant of myocardial salvage and mortality. Prompt revascularization (either by thrombolysis or primary angioplasty) is recommended for AMI patients but leads to deleterious effects called "reperfusion injury". Recently, ischemic postconditioning (PostC) efficiency has been reported in patients. However, its application during primary angioplasty is limited to patients admitted in angioplasty centers thereby excluding thrombolysed patients. In order to improve cardioprotection, it is necessary to define the time window for optimal cardioprotection and to develop new pharmacological strategies for AMI patients. Our work, using an in vivo mouse model of ischemia-reperfusion, shows that PostC efficiency is maintained if applied the first 30 minutes after the onset of reperfusion. Furthermore, we evaluated the therapeutic potential of peptide inhibitors targeting apoptosis, which is responsible for cell death during ischemia-reperfusion. As a proof-of-concept study, the cardioprotective effects of Tat-BH4 and Pip2b-BH4 peptidic constructs have been revealed in vitro and in vivo. The peptidic constructs targeting FAS-DAXX interaction, injected intravenously as a single bolus at the time of reperfusion, reduced by 50% both infarct size and apoptosis. These pharmacological tools have been patented due to their high therapeutic potential.
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Možná úloha proteinu DAXX v zástavě buněčného cyklu a buněčné senescenci / A potential role of DAXX in cell cycle arrest and cellular senescenceValášek, Ján January 2014 (has links)
Death domain-associated protein 6 (DAXX) is a multifunctional protein involved in diverse cellular processes. It acts as a histone chaperone or regulator of transcription and apoptosis, in which is its role quite controversial. DAXX also participates in regulation of cell DNA damage response (DDR). DAXX co-creates and stabilizes complex with Mdm2, which negatively regulates the stability of p53, an important tumor suppressor, which is a part of signalling node in the DDR. If DNA damage is not lethal for the cell and unables it to proliferate, the irreversible state of cell cycle called cellular senescence takes place. Under physiological conditions, induction of senescence can prevent the development of tumorigenesis. Therefore, the description of mechanisms involved in the induction of senescence has potential clinical significance. In my thesis, I aimed to determine changes in the level of DAXX protein in senescent cells and to characterize the manner of its regulation. In tumor cells MCF-7 and primary BJ fibroblasts, I observed decrease in DAXX protein level and its regulation. I tested the hypothesis according to which an increase in DAXX level before DNA damage canprevent induction of cellular senescence, or increase in its expression during senescence can cause recovery of cell proliferation....
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Identification of the NLS and NES of DaxxYang, Yi-Chin 30 August 2004 (has links)
SUMO is a small ubiquitin-like modifier. The fluorescent fused SUMO (active for sumoylation) localized in the nucleus, while C-terminal truncated SUMO (inactive for sumoylation) diffused in the cytoplasm. Daxx is a SUMO target protein, locates predominantly in the nucleus. It has been identified as a component of the PODs. During extracellular stimulation, Daxx could be recruited to the cytoplasm with the existence of Ask1. Therefore, it is a shuttle protein. Daxx should contain nuclear localization signal (NLS) and nuclear export signal (NES) motifs. To identify the NES and NLS motifs on Daxx, Daxx were truncated into four segments. Several amino acids on the predicted NES and NLS motifs were mutated. Our results showed that the truncated Daxx fragments D1 (containing NES) and D4 (containing NLS2) could be translocated into nucleus independently. However, either NES or NLS2 mutants disrupted their translocation into nucleus. It indicated that both NES and NLS2 motif of Daxx were involved in the nuclear transport. Nevertheless the co-transfection of SUMOs and Daxx showed that the interactions between SUMO active form and Daxx mutants and between inactive SUMO and Daxx wild type rescued the nuclear transport function of Daxx mutants and inactive SUMO. Therefore, SUMO may play a role in the nuclear transport of Daxx by either sumoylation or interaction with Daxx in cytoplasm, and Daxx may recruit inactive SUMOs into nucleus by interaction.
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Der Einfluss des kernkörperassoziierten Transkriptionsfaktors death-associated protein(Daxx)auf die Apoptose von rheumatoiden synovialen FibroblastenCinski, Antje 26 September 2007 (has links) (PDF)
Die rheumatoide Arthritis (RA) ist eine Autoimmunerkrankung mit bevorzugter Manifestation an den Gelenken. Die RA ist charakterisiert durch eine chronische, systemische Entzündung, eine abnormale zelluläre und humorale Immunantwort und eine synoviale Hyperplasie. Die Ursache der synovialen Hyperplasie ist noch nicht eindeutig geklärt, aber es wird eine veränderte oder unvollständig ablaufende Apoptose der synovialen Fibroblasten vermutet. Die vorliegende Arbeit untersucht die Rolle des Apoptosemodulators death associated protein (Daxx) in rheumatoiden synovialen Fibroblasten (RA-SF). Als erstes wurde die Expression von Daxx in RA-SF gegenüber Osteoarthrosefibroblasten (OA-SF) untersucht. Dabei konnte gezeigt werden, dass die OA-SF eine höhere Expression von Daxx auf mRNA und Proteinebene gegenüber RA-SF aufweisen. Im weiteren Verlauf erfolgte die Untersuchung der subzellulären Lokalisation von Daxx in RA-SF mittels konfokaler Laserscan-Mikroskopie (Immunfluoreszenz). Dabei zeigte sich, dass Daxx vorwiegend im Zellkern und nur zu einem geringen Anteil im Zytoplasma lokalisiert ist. Weiterhin zeigte Daxx eine Kolokalisation mit dem Promyeloischen Leukämie Protein (PML), welches ausschließlich im Zellkern lokalisiert ist. Nun erfolgten die Untersuchungen zur Rolle von Daxx in der Apoptose von RA-SF mittels RNA Interferenz (RNAi). Zu diesem Zweck wurden 3 verschiedene small interfering RNA (siRNA) synthetisiert, die unterschiedliche Abschnitte auf der mRNA von Daxx umfassen. Die Überprüfung der Effektivität der siRNA erfolgte auf mRNA Ebene mittels Quantitativer Real Time PCR(TaqMan®) und auf Proteinebene im Westernblot in HeLa-Zellen und RA-SF. Dabei zeigte die siRNA(454-472) die stärkste Hemmung. In den Untersuchungen zur Apoptose in HeLa-Zellen und RA-SF, nach der Hemmung von Daxx durch die siRNA, zeigte sich, dass eine stärkere Hemmung von Daxx zu einer verminderten Empfindlichkeit der HeLa-Zellen und RA-SF auf die FasL-induzierte Apoptose führt. Die RA-SF und Makrophagen der Deckzellschicht synthetisieren Zytokine wie den Tumor Nekrose Faktor a (TNFa). Diese Erkenntnis dient als Grundlage zur Untersuchung des Einflusses von TNFa auf die Expression von Daxx auf mRNA- und Proteinebene. Dabei zeigte sich eine konzentrationsabhängige Erhöhung der Expression von Daxx. Als letztes erfolgte die Untersuchung zur Apoptose nach der TNFa Stimulation. Hierbei zeigte sich eine Reduktion der Apoptose, die von der TNFa Konzentration abhängig war, wobei sich hohe Schwankungsbreiten bei der Konzentration von 10ng/ml TNFa zeigten. In dieser Arbeit wurde durch die Hemmung von Daxx mittels siRNA eine pro-apoptotische Funktion des Moleküls in RA-SF nachgewiesen. Diese Ergebnisse sollen der weiteren Identifizierung von Signalwegen in der Apoptose bei RA-SF dienen.
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Posttranslační modifikace adaptorového proteinu DAXX v buněčné odpovědi na genotoxický stres / Posttranslational modification of the adapter protein DAXX in the cellular response to genotoxic stressBražina, Jan January 2016 (has links)
Maintaining the chromosome continuity and complete genetic information in human cells is crucial for cell survival and the whole organism. It prevents life-threatening pathologies and preserves genetic continuity. However, cellular DNA is exposed to both endogenous and exogenous stress damaging its content and integrity. This stress activates mechanisms involving detection and repair of these damaged sites (DDR). One of the most serious types of DNA damage double-stranded breaks (DSB) occuring when both strands are severed. DSBs trigger wave of PTMs that regulate protein interactions, nuclear localization and catalytic activity of hundreds of proteins. Such modifications include acetylation, methylation, SUMOylation, ubiquitinylation and especially phosphorylation. The most important kinases involved in DDR kinases are ATM, ATR and DNA-PK. These kinases are activated immediately after the detection of the damaged area. DAXX (Death-associated protein 6) is an adapter and predominantly nuclear protein, which is involved in chromatin remodeling, gene expression modulation, antiviral response and depositing histone H3.3 variants into chromatin or telomeres. Daxx is essential for murine embryogenesis, since the homozygous deletion is lethal in E9.5-10. In 2006 a study mapping the substrates of kinases...
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Analýza buněčné signalizace zprostředkované adaptérovým proteinem Daxx / Analysis of cell signaling mediated by the adapter protein DaxxŠvadlenka, Jan January 2016 (has links)
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Der Einfluss des kernkörperassoziierten Transkriptionsfaktors death-associated protein(Daxx)auf die Apoptose von rheumatoiden synovialen FibroblastenCinski, Antje 17 April 2007 (has links)
Die rheumatoide Arthritis (RA) ist eine Autoimmunerkrankung mit bevorzugter Manifestation an den Gelenken. Die RA ist charakterisiert durch eine chronische, systemische Entzündung, eine abnormale zelluläre und humorale Immunantwort und eine synoviale Hyperplasie. Die Ursache der synovialen Hyperplasie ist noch nicht eindeutig geklärt, aber es wird eine veränderte oder unvollständig ablaufende Apoptose der synovialen Fibroblasten vermutet. Die vorliegende Arbeit untersucht die Rolle des Apoptosemodulators death associated protein (Daxx) in rheumatoiden synovialen Fibroblasten (RA-SF). Als erstes wurde die Expression von Daxx in RA-SF gegenüber Osteoarthrosefibroblasten (OA-SF) untersucht. Dabei konnte gezeigt werden, dass die OA-SF eine höhere Expression von Daxx auf mRNA und Proteinebene gegenüber RA-SF aufweisen. Im weiteren Verlauf erfolgte die Untersuchung der subzellulären Lokalisation von Daxx in RA-SF mittels konfokaler Laserscan-Mikroskopie (Immunfluoreszenz). Dabei zeigte sich, dass Daxx vorwiegend im Zellkern und nur zu einem geringen Anteil im Zytoplasma lokalisiert ist. Weiterhin zeigte Daxx eine Kolokalisation mit dem Promyeloischen Leukämie Protein (PML), welches ausschließlich im Zellkern lokalisiert ist. Nun erfolgten die Untersuchungen zur Rolle von Daxx in der Apoptose von RA-SF mittels RNA Interferenz (RNAi). Zu diesem Zweck wurden 3 verschiedene small interfering RNA (siRNA) synthetisiert, die unterschiedliche Abschnitte auf der mRNA von Daxx umfassen. Die Überprüfung der Effektivität der siRNA erfolgte auf mRNA Ebene mittels Quantitativer Real Time PCR(TaqMan®) und auf Proteinebene im Westernblot in HeLa-Zellen und RA-SF. Dabei zeigte die siRNA(454-472) die stärkste Hemmung. In den Untersuchungen zur Apoptose in HeLa-Zellen und RA-SF, nach der Hemmung von Daxx durch die siRNA, zeigte sich, dass eine stärkere Hemmung von Daxx zu einer verminderten Empfindlichkeit der HeLa-Zellen und RA-SF auf die FasL-induzierte Apoptose führt. Die RA-SF und Makrophagen der Deckzellschicht synthetisieren Zytokine wie den Tumor Nekrose Faktor a (TNFa). Diese Erkenntnis dient als Grundlage zur Untersuchung des Einflusses von TNFa auf die Expression von Daxx auf mRNA- und Proteinebene. Dabei zeigte sich eine konzentrationsabhängige Erhöhung der Expression von Daxx. Als letztes erfolgte die Untersuchung zur Apoptose nach der TNFa Stimulation. Hierbei zeigte sich eine Reduktion der Apoptose, die von der TNFa Konzentration abhängig war, wobei sich hohe Schwankungsbreiten bei der Konzentration von 10ng/ml TNFa zeigten. In dieser Arbeit wurde durch die Hemmung von Daxx mittels siRNA eine pro-apoptotische Funktion des Moleküls in RA-SF nachgewiesen. Diese Ergebnisse sollen der weiteren Identifizierung von Signalwegen in der Apoptose bei RA-SF dienen.
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Rôle de la chaperonne d'histone DAXX dans le maintien et l'établissement de l'hétérochromatine / Role of the histone chaperone DAXX in the maintenance and establishment of heterochromatinYettou, Guillaume 26 October 2012 (has links)
Le rôle fonctionnel des transcrits de l’hétérochromatine péricentromérique reste à ce jour largement incompris chez les eucaryotes supérieurs. Néanmoins, il a été montré que ces transcrits sont soumis à un contrôle très précis, fonction du cycle cellulaire. La régulation de la transcription est fortement contrôlée par la structure de la chromatine qui peut être modifiée localement en changeant la composition biochimique du nucléosome, notamment par l’utilisation des variantes d’histones. L’objectif de ma thèse a été de mieux comprendre le rôle de la protéine chaperonne d’histone DAXX et de sa variante d’histone H3.3 dans la régulation de la transcription des séquences répétées péricentromériques. Par la méthode de purification TAP-TAG, les partenaires spécifiques de DAXX ont été identifiés à partir d’extraits solubles nucléaires de fibroblastes embryonnaires murins. Ces analyses ont mis en évidence que CAF-1, classiquement associé à H3.1, et les facteurs de remodelage de la chromatine ATRX et CHD4 interagissent spécifiquement avec DAXX. Le rôle de ces protéines dans le contrôle de la transcription de l’hétérochromatine péricentromérique a ensuite été mis en évidence par une approche combinant l’interférence ARN et la Q-PCR. Enfin, les résultats suggèrent fortement que ces mécanismes de régulation ont lieu au niveau des corps nucléaires PML. L’ensemble de ces données montre qu’il existe une régulation spatio-temporel très fine de la structure de la chromatine régulant la transcription de l’hétérochromatine péricentromérique. / The functional role of pericentromeric heterochromatin transcripts remains largely unknown in higher eukaryotes. Nevertheless, it has been shown that these transcripts are subject to very precise control, depending on the cell cycle. Regulation of transcription is tightly controlled by chromatin structure that can be modified locally by changing the biochemical composition of the nucleosome, including the use of histone variants. The aim of my thesis was to better understand the role of the histone chaperone protein DAXX and its histone variant H3.3 in the regulation of transcription of pericentromeric repeats. By the method of TAP-TAG purification, DAXX specific partners were identified from soluble nuclear extracts of murine embryonic fibroblasts. These analyzes revealed that CAF-1, classically associated with H3.1, and the chromatin remodeling factors, ATRX and CHD4, specifically interact with DAXX. The role of these proteins in the control of transcription of pericentromeric heterochromatin was then highlighted by an approach combining RNAi and Q-PCR. Finally, the results strongly suggest that these regulatory mechanisms take place at PML nuclear bodies. Taken together, these data show that there is a spatio-temporal regulation of the fine structure of chromatin regulates transcription of pericentromeric heterochromatin.
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