XRCC1 Coordinates Disparate Responses and Multiprotein Repair Complexes Depending on the Nature and Context of the DNA Damage

XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLβ and PNK colocalize with XRCC1 in replication foci and that POLβ and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLβ and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLβ to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc

The Prevalence of TNFα-Induced Necrosis over Apoptosis Is Determined by TAK1-RIP1 Interplay

Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGFβ-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-α (TNFα) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNFα-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNFα-stimulated TNF-receptor I and also to inhibit the formation of TNFα-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIPL. The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation

Blockade of the LRP16-PKR-NF-κB signaling axis sensitizes colorectal carcinoma cells to DNA-damaging cytotoxic therapy.

Acquired therapeutic resistance by tumors is a substantial impediment to reducing the morbidity and mortality that are attributable to human malignancies. The mechanisms responsible for the dramatic shift between chemosensitivity and chemoresistance in colorectal carcinoma have not been defined. Here, we report that LRP16 selectively interacts and activates double-stranded RNA-dependent kinase (PKR), and also acts as scaffolds to assist the formation of a ternary complex of PKR and IKK beta, prolonging the polymers of ADP-ribose (PAR)-dependent nuclear factor kappa B (NF-kappa B) transactivation caused by DNA-damaging agents and confers acquired chemoresistance. We also identified a small molecule, MRS2578, which strikingly abrogated the binding of LRP16 to PKR and IKK beta, converting LRP16 into a death molecule and forestalling colon tumorigenesis. Inclusion of MRS2578 with etoposide, versus each drug alone, exhibited synergistic antitumor cytotoxicity in xenografts. Our combinatorial approach introduces a strategy to enhance the efficacy of genotoxicity therapies for the treatment of tumors.Peer reviewe

Role of poly(ADP-ribose) polymerases in the regulation of inflammatory processes

AbstractPARP enzymes influence the immune system at several key points and thus modulate inflammatory diseases. PARP enzymes affect immune cell maturation and differentiation and regulate the expression of inflammatory mediators such as cytokines, chemokines, inducible nitric oxide synthase and adhesion molecules. Moreover, PARP enzymes are key regulators of cell death during inflammation-related oxidative and nitrosative stress. Here we provide an overview of the different inflammatory diseases regulated by PARP enzymes

ZEB2 Mediates Multiple Pathways Regulating Cell Proliferation, Migration, Invasion, and Apoptosis in Glioma

BACKGROUND: The aim of the present study was to analyze the expression of Zinc finger E-box Binding homeobox 2 (ZEB2) in glioma and to explore the molecular mechanisms of ZEB2 that regulate cell proliferation, migration, invasion, and apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Expression of ZEB2 in 90 clinicopathologically characterized glioma patients was analyzed by immunohistochemistry. Furthermore, siRNA targeting ZEB2 was transfected into U251 and U87 glioma cell lines in vitro and proliferation, migration, invasion, and apoptosis were examined separately by MTT assay, Transwell chamber assay, flow cytometry, and western blot. RESULTS: The expression level of ZEB2 protein was significantly increased in glioma tissues compared to normal brain tissues (P<0.001). In addition, high levels of ZEB2 protein were positively correlated with pathology grade classification (P = 0.024) of glioma patients. Knockdown of ZEB2 by siRNA suppressed cell proliferation, migration and invasion, as well as induced cell apoptosis in glioma cells. Furthermore, ZEB2 downregulation was accompanied by decreased expression of CDK4/6, Cyclin D1, Cyclin E, E2F1, and c-myc, while p15 and p21 were upregulated. Lowered expression of ZEB2 enhanced E-cadherin levels but also inhibited β-Catenin, Vimentin, N-cadherin, and Snail expression. Several apoptosis-related regulators such as Caspase-3, Caspase-6, Caspase-9, and Cleaved-PARP were activated while PARP was inhibited after ZEB2 siRNA treatment. CONCLUSION: Overexpression of ZEB2 is an unfavorable factor that may facilitate glioma progression. Knockdown ZEB2 expression by siRNA suppressed cell proliferation, migration, invasion and promoted cell apoptosis in glioma cells

Mechanisms of NF-κB activation by genotoxic stress

Bei der zellulären DNA-Schadensantwort führt die Aktivierung des Transkriptionsfaktors NF-κB zur Induktion anti-apoptotischer Gene. Es wird angenommen, dass dieses Überlebensprogramm im Zusammenspiel mit Zellzyklus- Regulation und DNA-Reparaturvorgängen dafür sorgt, dass im Fall mäßiger DNA- Schädigung der zelluläre Lebenszyklus wieder aufgenommen werden kann. Im Rahmen der vorgelegten Dissertation wurde ein komplexes Signalnetzwerk aufgedeckt, das der NF-κB-Aktivierung durch DNA-Strangbrüche zugrunde liegt. Die zentralen Mediatoren zellulärer DNA-Schadensreaktionen PARP-1 und ATM induzieren zwei Signalachsen, die auf der Ebene der IKK-Aktivierung im Zytoplasma konvergieren. Nach Rekrutierung an DNA-Strangbrüche wird PARP-1 automodifiziert und ins Nukleoplasma freigesetzt. Daraufhin bildet das PARylierte Enzym einen transienten Komplex mit IKKγ, PIASy und ATM, wobei Protein-PAR-Wechselwirkungen eine stabilisierende Funktion wahrnehmen. Anschließend wird IKKγ durch PIASy mit SUMO1 modifiziert. Dieser Signalschritt ist für die Aktivierung des IKK-NF-κB-Systems erforderlich. Simultan wird ATM Ca2+-abhängig aus dem Zellkern exportiert und induziert nach Assoziation mit der Plasmamembran eine zweite Signalachse. Diese besteht aus einer Kette von Prozessen, die Ähnlichkeiten zu einigen klassischen NF-κB-Signalwegen, wie dem IL-1/Toll-Weg, aufweist. ATM bewirkt eine Aktivierung der Ubiquitin-Ligase TRAF6, die in Kooperation mit dem E2-Enzymkomplex Ubc13/Uev1A Lys63-verknüpfte Polyubiquitin-Ketten an sich selbst und eventuell an noch nicht identifizierten Signalkomponenten konjugiert. Nach der gegenwärtigen Vorstellung werden TAK1- und IKK-Komplexe an diese nicht-degradativen Ubiquitin-Polymere rekrutiert und in räumliche Nähe zu einander gebracht. Daraufhin kommt es zu einer trans-Autophosphorylierung und Aktivierung der Kinase TAK1, die für die Phosphorylierung und Aktivierung von IKKβ verantwortlich ist. IKKγ wird nach der Rekrutierung des IKK-Komplexes an Lys63-polyubiquitinierte Strukturen mono- und diubiquitiniert. Neben IKKβ- Phosphorylierung und IKKγ-Sumoylierung stellt diese Modifikation eine weitere Voraussetzung für die Aktivierung des IKK-Komplexes durch genotoxischen Stress dar. Da auch PARP-1 und PIASy neben ATM für Mono-/Diubiquitinierung von IKKγ erforderlich sind, stellt diese IKKγ-Modifikation den Konvergenzpunkt des ATM- und des PARP-1-ausgelösten Signalmoduls dar. Die präsentierten Ergebnisse ergeben ein neues Konzept der NF-κB-Aktivierung durch genotoxischen Stress. Neben der Identifikation neuer Schlüsselfaktoren des Signalwegs (PARP-1, TRAF6, TAB2, TAK1) konnten grundlegende Regulationsmechanismen dieser Kern- Zytoplasma-Signalkaskade aufgedeckt werden. Charakteristisch ist die Synthese von gerüstbildenden Polymeren (PAR und Lys63-Ubiquitin-Polymeren), an denen Multiproteinkomplexe assembliert werden (PARP-Signalosom und TAK1-IKK- Signalosom). In diesen supramolekularen Strukturen wird der Signalfluss integriert und an Enzyme weitergeleitet, die post-translationale Modifikationen an IKK-Untereinheiten vornehmen (IKKγ-Sumoylierung, IKKγ- Ubiquitinierung, IKKβ-Phosphorylierung). Diese Modifikationen beeinflussen sich zum Teil gegenseitig und tragen gemeinsam zur Aktivierung des IKK- Komplexes durch genotoxischen Stress bei.Activated as part of the cellular response to DNA-damage, the transcription factor NF-κB regulates the expression of anti-apoptotic genes. Thus, in case of moderate genotoxic stress, this survival program, in cooperation with cell cycle regulation and DNA repair, facilitates the resumption of cellular life cycle. The present thesis reveals a complex signaling network leading to NF-κB activation by DNA strand breaks. PARP-1 and ATM, as the central mediators of cellular responses to DNA damage, induce two signaling cascades, which converge on the activation of the IKK complex in the cytoplasm. Upon recruitment to DNA strand breaks, automodified PARP-1 is released into the nucleoplasm and forms a transient complex with IKKγ, PIASy and ATM. This signalosome is stabilized by protein-PAR-interactions and promotes PIASy mediated sumoylation of IKKγ, a modification necessary for the activation of the IKK-NF-κB system by genotoxic stress. Simultaneously, ATM is exported out of the nucleus in a Ca2+-dependent manner and induces the second identified signaling cascade at the plasma membrane. This pathway exhibits some characteristics of the classical NF-κB activation cascades, e.g. IL-1/Toll receptor signaling. ATM activates the ubiquitin ligase TRAF6, which functions in concert with the E2 enzyme complex Ubc13/Uev1A and conjugates Lys63-linked poly-ubiquitin chains to itself and possibly to still unidentified signaling factors. According to the current model, non-degradative ubiquitin polymers recruit TAK1 and IKK complexes, thereby bringing them into close proximity. Subsequently, TAK1 kinase is activated by trans-autophosphorylation, leading to phosphorylation and activation of IKKβ. Upon recruitment to Lys63-polyubiquitinated structures, IKKγ becomes mono- and diubiquitinated. Along with IKKβ phosphorylation and IKKγ sumoylation, this modification is prerequisite for the activation of the IKK complex by genotoxic stress. As in addition to ATM, PARP-1 and PIASy are also required for mono/diubiquitination of IKKγ, the ATM and the PARP-1 triggered signaling modules converge at the level of this IKKγ modification. The present work establishes a new concept of NF-κB activation by genotoxic stress. In addition to the identification of new key regulators (PARP-1, TRAF6, TAB2, TAK1) fundamental regulatory mechanisms of this nuclear-to-cytoplasmic signaling network could be uncovered. One hallmark is the synthesis of polymers with scaffolding function (PAR and Lys63-ubiquitin polymers), which facilitate the assembly of multiprotein complexes (PARP signalosome and TAK1-IKK signalosom, respectively). Within these supramolecular structures, the signaling information is integrated and transduced to enzymes, which post-translationally modify IKK subunits (IKKγ sumoylation, IKKγ ubiquitination and IKKβ phosphorylation). These modifications are in part interdependent and contribute cooperatively to the activation of the IKK complex by genotoxic stress

I was luckier than most.

Regina Cooper tells about her recollection of ‘Anschluss’ (March 1938), when Austria was annexed by Nazi Germany; ‘Kristallnacht’ (November 1939); her flight to England; her immigration to the United States; and her reunion with her parents.Donated by Regina Cooper via AHCRegina Cooper née Stielmann was born 1927 in Vienna, Austria. She emigrated with the help of Kindertransport to London and from there to the United States, where she arrived in 1943. She settled in New York City.Austrian Heritage Collectiondigitize

A cytoplasmic ATM-TRAF6-cIAP1 module links nuclear DNA damage signaling to ubiquitin-mediated NF-κB activation

As part of the genotoxic stress response, cells activate the transcription factor NF-κB. The DNA strand break sensor poly(ADP-ribose)-polymerase-1 (PARP-1) and the kinase ataxia telangiectasia mutated (ATM) act as proximal signal mediators. PARP-1 assembles a nucleoplasmic signalosome, which triggers PIASy-mediated IKKγ SUMOylation. ATM-dependent IKKγ phosphorylation and subsequent ubiquitination were implicated to activate the cytoplasmic IκB kinase (IKK) complex by unknown mechanisms. We show that activated ATM translocates in a calcium-dependent manner to cytosol and membrane fractions. Through a TRAF-binding motif, ATM activates TRAF6, resulting in Ubc13-mediated K63-linked polyubiquitin synthesis and cIAP1 recruitment. The ATM-TRAF6-cIAP1 module stimulates TAB2-dependent TAK1 phosphorylation. Both nuclear PARP-1- and cytoplasmic ATM-driven signaling branches converge at the IKK complex to catalyze monoubiquitination of IKKγ at K285. Our data indicate that exported SUMOylated IKKγ acts as a substrate. IKKγ monoubiquitination is a prerequisite for genotoxic IKK and NF-κB activation, but also promotes cytokine signaling. Copyright © 2011 Elsevier B.V. All rights reserved