Etablierung der Interaktion des viralen Onkoproteins LMP1 mit den zellulären Signalproteinen der TRAF-Proteinfamilie als Zielstruktur für Inhibitoren

Das Epstein-Barr Virus (EBV) ist mit einer Reihe von lebensbedrohlichen Krankheiten assoziiert. Dazu zählen unter anderem Nasopharynxkarzinome, Hodgkin-Lymphome und lymphoproliferative Erkrankungen nach Organtransplantationen. Dennoch gibt es bisher keinen wirksamen Therapieansatz, der sich spezifisch mit der Rolle von EBV in diesen malignen Erkrankungen auseinandersetzt. Das latente Membranprotein 1 (LMP1) ist das primäre Onkogen von EBV und essenziell für die Transformation von B-Zellen durch das Virus. Für eine effiziente Transformation von Zellen ist die Aktivierung verschiedener zellulärer Signalwege durch LMP1 notwendig. LMP1 besitzt jedoch keine enzymatische Aktivität und die Induktion der Signalwege ist somit abhängig von der Rekrutierung verschiedener zellulärer Adapterproteine. Die Ausbildung der notwendigen Signalkomplexe wird über zwei C-terminale Aktivierungs-Regionen (CTAR1 und CTAR2) vermittelt. Verschiedene Mitglieder der Tumornekrosefaktor (TNF)-Rezeptor-assoziierten Faktoren (TRAF)-Protein-Familie spielen bei der Induktion der Signalwege durch diese beiden CTAR-Domänen eine zentrale Rolle. Nach grundlegenden Protein-Protein-Interaktionsstudien zwischen LMP1 und rekombinanten TRAF-Proteinen wurde hier die Interaktion zwischen TRAF2 und LMP1 als Zielstruktur für Inhibitoren vorgestellt. TRAF2 ist essenziell für die Aktivierung des NF-κB-Signalweges durch die CTAR1-Domäne und somit für das Überleben EBV-transformierter Zellen. Die Bindung von TRAF2 an LMP1 wurde biochemisch näher charakterisiert und die gewonnen Erkenntnisse verwendet, um ein System zu etablieren, mit dem Inhibitoren gegen den Komplex aus LMP1 und TRAF2 identifiziert werden können. Dieses ELISA-basierte System erfüllt die Anforderungen, die allgemein an hochdurchsatzfähige Systeme gestellt werden. In einem Pilotscreen einer Bibliothek mit Naturstoffen wurden Substanzen identifiziert, die die Bindung von TRAF2 an LMP1 in vitro inhibierten. Die potenteste Substanz inhibierte die Interaktion von TRAF2 und LMP1 mit einem IC50 von 8 µM in diesen in vitro Studien. Weiterhin zeigte diese Substanz eine spezifische biologische Wirkung auf die Vitalität von EBV-transformierten B-Zellen. Zusätzlich konnte in den Protein-Protein-Interaktionsstudien zwischen den verschiedenen TRAF-Proteinen und LMP1 erstmals eine direkte Bindung von TRAF6 an LMP1 gezeigt werden. Entgegen der bisherigen Modellvorstellung, nach der TRAF6 indirekt über Adapterproteine an LMP1 gebunden wird, konnte hier gezeigt werden, dass TRAF6 direkt an die LMP1-Sequenz P379VQLSY innerhalb der CTAR2-Domäne bindet. Diese Sequenz ist essenziell für die Aktivierung verschiedener TRAF6-abhängiger Signalwege durch die CTAR2-Domäne. Auf der Oberfläche von TRAF6 wird die Bindung an LMP1 durch dieselbe Bindetasche vermittelt, über die auch die Interaktion mit zellulären Rezeptoren stattfindet. Diese direkte Interaktion zwischen LMP1 und TRAF6 ist wichtig für die Aktivierung des NF κB-Signalweges durch die CTAR2-Domäne. TRAF6-Mutanten, die nicht mehr in der Lage waren, mit LMP1 zu interagieren, waren ebenfalls nicht mehr dazu fähig, die Induktion von NF κB-Signalen durch die CTAR2-Domäne von LMP1 in embryonalen TRAF6-/- Mausfibroblasten wiederherzustellen. Ebenfalls konnte neben der direkten Bindung von TRAF6 an LMP1 hier eine weitere neue Protein-Protein-Interaktion für TRAF6 beschrieben werden. TRAF6 bindet direkt an das TNF-Rezeptor-assoziierte Todesdomänenprotein (TRADD). Die Interaktion zwischen TRAF6 und TRADD unterscheidet sich jedoch von der Bindung anderer TRAF-Proteine an TRADD. Die in vitro Studien zeigten, dass TRAF6 in der Lage ist, sowohl mit Teilen des N-Terminus, als auch mit Teilen des C-Terminus von TRADD zu interagieren. Diese bisher nicht beschriebene Art der direkten Interaktion von TRAF6 mit TRADD eröffnet neue Einblicke in den Aufbau des LMP1-Signalkomplexes

A central role of IKK2 and TPL2 in JNK activation and viral B-cell transformation

IκB kinase 2 (IKK2) is well known for its pivotal role as a mediator of the canonical NF-κB pathway, which has important functions in inflammation and immunity, but also in cancer. Here we identify a novel and critical function of IKK2 and its co-factor NEMO in the activation of oncogenic c-Jun N-terminal kinase (JNK) signaling, induced by the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). Independent of its kinase activity, the TGFβ-activated kinase 1 (TAK1) mediates LMP1 signaling complex formation, NEMO ubiquitination and subsequent IKK2 activation. The tumor progression locus 2 (TPL2) kinase is induced by LMP1 via IKK2 and transmits JNK activation signals downstream of IKK2. The IKK2-TPL2-JNK axis is specific for LMP1 and differs from TNFα, Interleukin-1 and CD40 signaling. This pathway mediates essential LMP1 survival signals in EBV-transformed human B cells and post-transplant lymphoma, and thus qualifies as a target for treatment of EBV-induced cancer

Bioinspired Design of Lysolytic Triterpenoid-Peptide Conjugates that Kill African Trypanosomes.

Humans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). Active TLF-ingredient is the primate-specific apolipoprotein L1 (ApoL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies only two are able to counteract the activity of ApoL1, which illustrates its evolutionary optimized design and trypanocidal potency. Here we ask the question whether a synthetic (syn)TLF can be synthesized using the design principles of the natural TLF-complexes but relying on different chemical building blocks. We demonstrate the stepwise development of triterpenoid-peptide conjugates, in which the triterpenoids act as a cell binding, uptake and lysosomal transport-moduls and the synthetic peptide GALA as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis demonstrating proof-of-principle for the bioinspired, forward-design of a synTLF

The germinal center kinase TNIK is required for canonical NF-κB and JNK signaling in B-cells by the EBV oncoprotein LMP1 and the CD40 receptor.

The tumor necrosis factor-receptor-associated factor 2 (TRAF2)- and Nck-interacting kinase (TNIK) is a ubiquitously expressed member of the germinal center kinase family. The TNIK functions in hematopoietic cells and the role of TNIK-TRAF interaction remain largely unknown. By functional proteomics we identified TNIK as interaction partner of the latent membrane protein 1 (LMP1) signalosome in primary human B-cells infected with the Epstein-Barr tumor virus (EBV). RNAi-mediated knockdown proved a critical role for TNIK in canonical NF-κB and c-Jun N-terminal kinase (JNK) activation by the major EBV oncoprotein LMP1 and its cellular counterpart, the B-cell co-stimulatory receptor CD40. Accordingly, TNIK is mandatory for proliferation and survival of EBV-transformed B-cells. TNIK forms an activation-induced complex with the critical signaling mediators TRAF6, TAK1/TAB2, and IKKβ, and mediates signalosome formation at LMP1. TNIK directly binds TRAF6, which bridges TNIK's interaction with the C-terminus of LMP1. Separate TNIK domains are involved in NF-κB and JNK signaling, the N-terminal TNIK kinase domain being essential for IKKβ/NF-κB and the C-terminus for JNK activation. We therefore suggest that TNIK orchestrates the bifurcation of both pathways at the level of the TRAF6-TAK1/TAB2-IKK complex. Our data establish TNIK as a novel key player in TRAF6-dependent JNK and NF-κB signaling and a transducer of activating and transforming signals in human B-cells

TNIK is essential for JNK activation by LMP1.

<p>(A) HEK293 cells were transfected in 6-well plates with siRNA against human TNIK (siTNIK) or non-targeting control siRNA (siCTR). Subsequently, the cells were co-transfected with 1 µg of HA-JNK1 and 1 µg of HA-LMP1 wildtype or HA-LMP1Δ194–386 lacking the LMP1 signaling domain, as indicated. HA-JNK1 was immunoprecipitated from cell lysates and immunocomplex kinase assays were performed using recombinant GST-c-Jun as a substrate. Equal HA-JNK1 immunoprecipitation was confirmed by the anti-JNK1 antibody. TNIK downregulation and LMP1 expression was monitored in cell lysates using anti-TNIK and anti-LMP1 (CS1-4) antibodies. The null control construct LMP1Δ194–386 cannot be detected by the CS1-4 antibody because all CS1-4 epitopes are located within the LMP1 signaling domain. Quantification of four independent experiments ± standard deviations, given as x-fold inductions: lane 1, 1.15±0.32; lane 2, 1.48±0.6; lane 3, 1.0±0.0; lane 4, 3.23±1.03. (B) The knockdown of TNIK in lymphoblastoid cells blocks the JNK pathway. EREB2-5 B-cells were treated with Accell siRNA against human TNIK or non-targeting siRNA for 72 h. Cell lysates were analyzed by immunoblotting using anti-TNIK, anti-phospho-JNK, anti-JNK1, and anti-LMP1 (CS1-4) antibodies. <i>n</i> = 3.</p

TNIK forms a dynamic signaling complex with TAK1, TAB2, and IKKβ upon LMP1 activation.

<p>(A) TAK1 interacts with TNIK independent of LMP1. HEK293 cells were transfected in 15 cm culture dishes with 5 µg of HA-TNIK and 2 µg of Flag-TAK1 vectors, both in the presence and absence of 5 µg pSV-LMP1, as indicated. HA-TNIK was immunoprecipitated with the anti-HA (12CA5) antibody. Immunoprecipitations were analyzed using anti-TNIK and anti-TAK1 antibodies. Cell lysates were stained with anti-TNIK, anti-TAK1, and anti-LMP1 (CS1-4) antibodies. <i>n</i> = 3. (B) TAK1 binds to the GCKH and intermediate domains of TNIK. HEK293 cells were transfected in 10 cm cell culture dishes with 2 µg of the indicated HA-TNIK constructs and 1 µg of Flag-TAK1 vector. HA-TNIK constructs were immunoprecipitated with the anti-HA (12CA5) antibody. Immunoprecipitations and lysates were analyzed with anti-HA (3F10) and anti-TAK1 antibodies, as indicated. Asterisks indicate TNIK proteins. Apparent molecular masses are given in kDa. <i>n</i> = 4. (C) LMP1 induces the interaction between TNIK and TAB2. HEK293 cells were transiently transfected in 15 cm culture dishes with 5 µg of HA-TAB2 and Flag-TNIK vectors in the presence or absence of 5 µg pSV-LMP1 as indicated. Flag-TNIK was immunoprecipitated using the anti-Flag (6F7) antibody. The following antibodies were used for immunoblotting: anti-Flag (6F7), anti-TAB2, anti-LMP1 (1G6-3). <i>n</i> = 3. (D) TNIK is essential for TAK1 interaction with LMP1. HEK293 cells were transfected in 15 cm culture dishes with 5 µg Flag-TAK1, 3 µg LMP1, and 7 µg each of shTNIK or shCTR vectors, as indicated. Cells were lysed 48 h post-transfection, LMP1 was immunoprecipitated using the anti-LMP1 (1G6-3) antibody, and co-precipitation of Flag-TAK1 was analyzed by immunoblotting for TAK1. TNIK knockdown and Flag-TAK1 expression was verified in total cell lysates. <i>n</i> = 3. (E) TNIK mediates the interaction between TAB2 and LMP1. HEK293 cells were transfected with the indicated vectors as described in (D). LMP1 was precipitated from cell lysates using the anti-LMP1(1G6-3) antibody. Immunoprecipitations and lysates were analyzed with the indicated antibodies. <i>n</i> = 2. (F) LMP1 induces the recruitment of IKKβ to the TNIK complex. HEK293 cells were transfected in 10 cm culture dishes with 2 µg HA-TNIK, 2 µg Flag-IKKβ, and 3 µg pSV-LMP1 as indicated. HA-TNIK was immunoprecipitated using the anti-HA (12CA5) antibody and immunocomplexes were analyzed by immunoblotting using anti-HA (12CA5) and anti-Flag (M2) antibodies. Expression of HA-TNIK, Flag-IKKβ, and LMP1 was detected in cell lysates with the anti-HA (12CA5), anti-Flag (M2), and anti-LMP1 (1G6-3) antibodies. <i>n</i> = 3. (G) TNIK signaling complex containing LMP1, TRAF6, TAK1, TAB2, and IKKβ in EBV-transformed lymphoblastoid cells. Endogenous TNIK was immunoprecipitated from LCL 721 cells with the anti-TNIK antibody. An unrelated mouse isotype IgG was used for control precipitation (IsoG). As indicated, immunoprecipitations and lysates were analyzed by immunoblotting using the anti-TNIK, anti-TRAF6 (H-274), anti-LMP1 (1G6-3), anti-TAK1, anti-TAB2, and anti-IKKβ antibodies. <i>n</i> = 2.</p

TNIK mediates proliferation and survival in EBV-transformed human B-cells.

<p>(A) The knockdown of TNIK inhibits proliferation of LCLs. EREB2-5 cells were seeded in the presence of Accell siRNA targeting TNIK or non-targeting siRNA. Cell proliferation was monitored at the indicated times by MTT conversion. Shown are the results of one representative experiment in triplicates of three independent experiments. (B) Apoptosis induction by TNIK knockdown in LCLs. EREB2-5 cells were incubated with Accell siTNIK or siCTR, as indicated. Apoptosis was monitored after 3 d in the presence of siRNA by co-staining of the cells with Cy5-labeled annexin V (AnV-Cy5) and propidium iodide (PI). The population of intact cells within the lymphocyte gate in the forward scatter (FSC)/sideward scatter (SSC) plot (left panels) was strongly reduced after the knockdown of TNIK. The gated cells were then analyzed for apoptosis rates, indicated by PI−/AnV-Cy5+ staining (right panels). The graph shows mean values of PI−/AnV-Cy5+ percentages of three independent experiments ± standard deviations; two-tailed Student's <i>t</i> test. *<i>p</i> = 0.033.</p