Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1)

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.     

Immunity in plants and animals: common ends through different means using similar tools

A comparative approach is potentially useful for understanding the role of mammal innate immunity role in stimulating adaptive immunity as well as the relationship between these two types of immune strategies. Considerable progress has been made in the elucidation of the co-ordinated events involved in plant perception of infection and their mobilisation of defence responses. Although lacking immunoglobulin molecules, circulating cells, and phagocytic processes, plants successfully use pre-formed physical and chemical innate defences, as well as inducible adaptive immune strategies. In the present paper, we review some shared and divergent immune aspects present in both animals and plants.     

Aurora A kinase activation: Different means to different ends

Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation.

IntroductionDuring mitosis, almost 30% of the proteome is modified by the transfer of phosphate to serine, threonine, or tyrosine residues. These phosphorylation events are responsible for the profound transformation of cellular architecture and physiology that occurs as cells progress through mitosis. Pivotal protein kinases responsible for the massive increase in protein phosphorylation as cells transit into mitosis include Aurora A kinase (AURKA), Aurora B kinase, Polo-like kinase 1 (Plk1), and Cyclin-dependent kinase 1 (Cdk1)/Cyclin A/B complexes. Their precise and coordinated activation critically defines the G2-M transition.Overexpression and aberrant activation of AURKA have been functionally linked to oncogenic transformation through centrosome amplification, aneuploidy, and chromosomal instability. Beyond its pivotal role in mitotic cell division, AURKA has numerous nonmitotic functions in tumorigenesis. AURKA thus represents a critical “druggable target” in cancer, controlling key oncogenic pathways associated with drug resistance and poor patient outcome.AURKA activation is unexpectedly complex, and a number of different mechanisms have been described, including autophosphorylation of its activation segment and binding to a variety of allosteric modulators, which recruit and locally activate AURKA at specific subcellular localizations (Fig. 1). Recent findings show that these allosteric modulators activate AURKA through surprisingly distinct mechanisms, each acting at different subcellular locations to trigger a unique event in response to different upstream signals. We summarize current views of these activation mechanisms and speculate on the reasons underlying this complexity.Open in a separate windowFigure 1.Model of AURKA activation during cell cycle progression. Left panel: AURKA is activated during the G2/M transition by binding to Bora phosphorylated on its M3 motif by Cyclin A-Cdk1 (CycA-Cdk1). Phospho-Bora (pBora) binds unphosphorylated inactive AURKA (in gray, N and C denote the N-lobe and the C-lobe, respectively) via its M1, M2 (dark blue), and phospho-M3 (green) motifs. Binding of phospho-Bora turns on the catalytic activity of AURKA (red) by substituting in trans the phosphoregulatory site on Thr288, leading to mitotic entry. P denotes phosphate. Middle panel: AURKA is activated at centrosomes via Cep192-dependent oligomerization and oxidation of Cys290 by ROS. NEDB, nuclear envelope breakdown. Right panel: AURKA is activated at spindle microtubules by Tpx2 binding through M1 and M2 motifs and by autophosphorylation at Thr288.Autophosphorylation of the activation segment and binding to the allosteric regulator Targeting protein for Xklp2 (Tpx2) synergize to locally activate AURKA at microtubulesIn eukaryotes, the transfer of phosphate from ATP to protein substrates is mediated by the protein kinase domain, a bilobal catalytic entity. The protein kinase domain possesses a complicated structure, with many flexible parts but a highly restricted catalytic mechanism (i.e., there is only one way to transfer phosphate). This affords great opportunity for the diversification of how each kinase turns on and off (Endicott et al., 2012). Like the majority of eukaryotic protein kinases, AURKA is regulated by phosphorylation of a conserved residue, Thr288, within a flexible element of the kinase domain termed the activation segment. This event leads to a reorganization of the active site that is required, but not sufficient, for full catalytic activation. This is in sharp contrast to many other protein kinases, where phosphorylation of the activation segment is sufficient for maximal catalytic activation. Similar to the closely related AGC family kinases, AURKA has evolved a dependency for its full activation on the binding of an allosteric modulator to its smaller N-terminal kinase lobe (Leroux et al., 2018). This event positions or stabilizes structural elements in the kinase active site that are not sufficiently aligned by activation segment phosphorylation alone. For most AGC family kinases, such as the exemplars PKA and AKT, the allosteric modulator represents a linear peptide sequence contained within the protein kinase itself, but distal to the protein kinase domain. In contrast, in the case of AURKA, the allosteric modulator is presented by an entirely separate protein.The best characterized allosteric modulator of AURKA is the microtubule-binding protein Txp2. Upon nuclear envelope breakdown, Tpx2 is released by RAN-GTP from importins, which then allows it to concurrently recruit and activate AURKA at microtubules to promote mitotic spindle assembly. Tpx2 uses its first N-terminal 43 amino acids to activate AURKA, in a manner synergistic with activation segment phosphorylation, by binding across the N-lobe of the AURKA kinase domain (Fig. 1). Notably, in the absence of Tpx2 binding and activation segment phosphorylation, AURKA retains marginal but detectable protein kinase activity. Binding of Tpx2 alone boosts AURKA catalytic function modestly (15-fold) while autophosphorylation alone boosts catalytic function substantially (157-fold). However, the action of both events translates into a 448-fold enhancement in activity relative to the fully repressed state (Dodson and Bayliss, 2012). This coordination of maximal activation with the recruitment of AURKA to microtubules may serve a double duty to minimize spurious phosphorylation of proteins elsewhere in the cell.Autophosphorylation and binding to Tpx2 trigger conformational changes in AURKA that are sufficiently large to be probed by time-resolved fluorescence energy transfer approaches (Ruff et al., 2018). Time-resolved fluorescence energy transfer revealed that the activation segment of AURKA adopts a wide range of conformations in solution. Notably, binding of Tpx2 to AURKA locks an inward conformation of a catalytic element termed the DFG motif (the Asp in the Asp-Phe-Gly motif coordinates a magnesium ion required for ATP binding) by rigidifying an inherently flexible helix αC (Ruff et al., 2018). In contrast, Thr288 phosphorylation promotes a large conformational change in the activation segment that enables the binding of peptide substrates. Both Tpx2 binding and autophosphorylation are required for AURKA function at microtubules. This transient “doubly activated” form of AURKA is not detectable at spindle microtubules in normal cells due to the action of the AURKA-directed protein phosphatase 6 (PP6), which specifically dephosphorylates Tpx2-bound AURKA on the activation segment.Phospho-Bora activates unphosphorylated AURKA in the cytoplasm to trigger mitotic entryIn a manner thematically similar to how Tpx2 binding and AURKA autophosphorylation synergize to activate AURKA at microtubules, recent work revealed that a phosphorylated form of Bora activates cytoplasmic AURKA during mitotic commitment (Tavernier et al., 2021).Commitment to mitosis is tightly coordinated with DNA replication to preserve genome integrity. Commitment is achieved by a tightly choreographed biochemical tug-of-war between mitotic kinases and phosphatases (PPases). To this end, AURKA activates Plk1 by phosphorylating its activation segment at Thr210. In turn, Plk1 promotes activation of the Cdk1/Cyclin B complex by phosphorylating both negative and positive regulators of Cdk1 to trigger mitotic entry. As AURKA lies at the top of this mitotic kinase cascade, the key question that arises is how is AURKA initially activated in G2?As noted above, AURKA can autophosphorylate its own activation segment at Thr288, but this form of the enzyme is rapidly dephosphorylated by counteracting PPases in G2. As dephosphorylation maintains AURKA in an inactive state, how then does AURKA overcome the repressive effect of PPases to activate Plk1?AURKA activation during mitotic commitment is critically dependent on the evolutionarily conserved protein Bora, following its own phosphorylation on a key regulatory site on Ser112. This event is essential for the phosphorylation of Plk1 on Thr210 by AURKA in vitro and for timely mitotic entry in vivo, both in Xenopus egg extracts (Vigneron et al., 2018) and in human cells (Tavernier et al., 2021). Remarkably, phospho-Bora binds to and potently activates AURKA lacking phosphorylation of its activation segment, suggesting the possibility that the phosphate on S112 of Bora may physically and/or functionally substitute for the phosphorylated activation segment on AURKA.Dissection of how Bora binds AURKA revealed at least two motifs in Bora, denoted M1 and M2, with weak similarity to AURKA-binding elements in Tpx2^1–43. Both motifs are required for the binding and activating function of Bora on AURKA, and notably, the essential Ser112 phospho-regulatory site (in the sequence Pro-Ser-Pro, denoted motif M3) lies immediately C-terminal to binding motif M2. By analogy to the mechanism of action of Tpx2, Bora motif M1 likely binds in an extended manner parallel to the top surface of helix αC, whereas motif M2 adopts a helical conformation and binds parallel to the bottom surface of helix αC. As Bora motif M3 is immediately adjacent to motif M2, this binding mode would orient the Ser112 phospho-moiety of motif M3 in close proximity to a constellation of positively charged residues that normally engage the phosphate moiety of the phosphorylated activation segment of AURKA (Fig. 1; Tavernier et al., 2021). This mode of action elegantly allows phospho-Bora to allosterically activate AURKA during mitotic commitment, when AURKA itself is catalytically repressed by dephosphorylation. The precise atomic details of how phospho-Bora binds and activates AURKA and whether other protein kinases use analogous mechanisms for activation remain to be determined.Since phosphorylation of Bora Ser112 is essential for its ability to activate AURKA and commit cells to mitosis, the upstream kinase responsible for this regulatory event represents a critical component of the AURKA activation puzzle. This function is performed by Cyclin A–Cdk1, which is active in S-G2 and known to promote mitotic entry. Consistent with this model, Bora phosphorylated on S112 is sufficient to promote mitotic commitment in Xenopus egg extracts depleted of Cyclin A (Vigneron et al., 2018). In human cells, Cyclin A–Cdk1 is confined to the nucleus during S phase, but at the S/G2 transition it is abruptly exported to the cytoplasm, allowing it to phosphorylate Bora (Silva Cascales et al., 2021). As such, Bora acts as a bridge linking Cyclin A–Cdk1 activity to the activation of the mitotic kinase cascade. Why phospho-Bora can persist under conditions that disfavor AURKA activation by autophosphorylation remains an open question worthy of further investigation. Possibilities include that the phospho-Ser112 residue is a suboptimal PPase substrate or that it is protected from dephosphorylation by cis-activating factors.Redox regulation of AURKA during mitosisRecent work indicates that AURKA activity is also regulated by oxidative signaling with both stimulatory and inhibitory outcomes. While autophosphorylation of AURKA on Thr288 is largely neutralized in the cytoplasm and at spindle microtubules by counteracting PPases, it is readily detected at centrosomes. Centrosomal AURKA autoactivation is stimulated as a consequence of Cep192-mediated oligomerization and AURKA autophosphorylation, but the underlying mechanism was poorly understood. New studies reveal that oxidative modification of a conserved cysteine residue, Cys290, located in the activation segment of the kinase domain promotes AURKA autophosphorylation during mitosis when AURKA is oligomerized.While biochemical studies using purified proteins in the absence of an oligomerizing agent revealed that oxidative modification of Cys290 inhibited AURKA kinase activity (Byrne et al., 2020; Tsuchiya et al., 2020), cell treatment with oxidizing agents such as H₂0₂ increased AURKA phosphorylation on Thr288 (Wang et al., 2017; Tsuchiya et al., 2020). This increase in Thr288 phosphorylation was accompanied by dimerization of AURKA in a manner sensitive to reducing agents such as DTT. This result hinted that disulfide bond formation between AURKA monomers might be involved in promoting AURKA trans-autophosphorylation. Consistent with this hypothesis, a crystal structure of an AURKA kinase domain obtained under disulfide bond–promoting conditions revealed a face-to-face dimer orientation of the kinase domain stabilized by a Cys290–Cys290 disulfide bond (Lim et al., 2020). In this configuration, the active site of the AURKA kinase domain adopts a productive conformation predicted to support substrate phosphorylation. Given the inherent flexibility of the activation segment itself, this face-to-face configuration of the kinase domain was also predicted to support AURKA trans-autophosphorylation on Thr288. Follow-up biochemical studies proved that the Cys290–Cys290 disulfide–linked configuration of the AURKA kinase domain is indeed compatible with trans-autophosphorylation on Thr288.An interesting feature of the Cys290-dependent activation mechanism of AURKA is the requirement for Cep192. Presumably, the ability of Cep192 to recruit and oligomerize AURKA favors the formation of the Cys290–Cys290 disulfide bond between kinase domains (Fig. 1). In support of this model, oxidation-induced Cys290–Cys290 disulfide cross-linking of AURKA could also be recapitulated in Xenopus extracts by the addition of bivalent antibodies directed at AURKA.At subcellular locations beyond centrosomes, where AURKA is not dimeric, oxidation of the activation segment would be expected to have an opposite effect on protein kinase activity. For instance, a crystal structure of monomeric AURKA covalently bound to Coenzyme A (CoAlation), a major regulator of cellular metabolism that contains both nucleotide and thiol moieties, revealed that AURKA CoAlation robustly inhibits kinase activity (Tsuchiya et al., 2020) through an ATP-competitive mechanism. Competitive binding is achieved by the nucleotide moiety of Coenzyme A engaging the nucleotide-binding pocket of ATP, while the reactive pantetheine thiol moiety forms a disulfide bond with Cys290. This dual anchoring of Coenzyme A to AURKA imparts not only affinity but also specificity toward kinase inhibition. Supporting the possibility that Coenzyme A can exert a potent inhibitory effect on AURKA under physiological conditions, microinjection of CoA into mouse oocytes caused abnormal spindles and chromosome misalignment, phenotypes typically observed upon AURKA inactivation.Interestingly, when bound to AURKA, Tpx2 exerts a protective effect against inhibition by CoAlation. Given that Bora is predicted to bind AURKA similar to Tpx2 (Tavernier et al., 2021), this could allow Bora to also protect AURKA from inhibition by CoAlation. Together, these results suggest that each distinct cellular pool of AURKA will respond differently to oxidation signals.Reactive oxygen species (ROS) are emerging as important signaling molecules. ROS and oxidative stress have been shown to increase during G2 and M phases in an otherwise unperturbed asynchronous cell cycle (Patterson et al., 2019), suggesting that oxidative modification of biomolecules, including AURKA, might regulate mitotic progression. Likewise, H₂O₂ locally released by mitochondria, where a pool of AURKA has been recently shown to localize (Bertolin et al., 2018), is implicated in symmetry breaking and polarity establishment in early Caenorhabditis elegans embryos (De Henau et al., 2020). It will be particularly exciting to determine whether AURKA, which also plays a role in setting up embryo polarity, is regulated by redox signaling in this specific context.Concluding remarksAURKA is activated by a growing list of mechanisms, with each acting at specific stages of the cell cycle and subcellular location. The ability to monitor which specific mechanism is at play at any one time in vivo presents a particular challenge. The activation state of AURKA is often measured by the use of a phospho-specific antibody targeting the phosphorylated Thr288 epitope. However, this has limited effectiveness to detect AURKA activated by Tpx2 at spindle microtubules because of the transient nature of the phospho-Thr288 epitope at this location. Furthermore, in the case of cytoplasmic AURKA activation by Bora, phosphorylation at Thr288 is not required for kinase activation. A live fluorescence energy transfer sensor has been reported for the phosphorylation status of AURKA on Thr288 that detects conformational changes induced by Thr288 phosphorylation rather than the phosphorylation motif itself (Bertolin et al., 2016). If the binding of Tpx2 to phosphorylated AURKA and the binding of phospho-Bora to dephosphorylated AURKA induces similar conformational changes to those induced by autophosphorylation, then this could represent a more generally applicable assay for monitoring the activation state of AURKA.Why is the activation of AURKA so complex? We speculate that the major reason for this complexity is related to kinase action at distinct times and in spatially distinct locations (Fig. 1). Bora acts in the cytoplasm before mitotic entry, and Cep192 acts at the centrosome before and likely after mitotic entry, whereas Tpx2 acts on spindle microtubules after mitotic entry. Thus, the allosteric regulators and their own upstream controllers (e.g., Cyclin A/Cdk1 for Bora) direct AURKA activity to execute distinct functions. In some ways, this complexity of activation represents a different solution than the one adopted by Plk1 or Protein Phosphatase 1 (PP1), which also acts at distinct time points and subcellular locations. Both Plk1 and PP1 employ docking motifs that are post-translationally controlled to dictate their time and sites of action, as well as their substrate specificity. As deeper insights are gained into the control of mitotic kinases and PPases, it will be intriguing to see what additional solutions have evolved to address the challenge of temporally and spatially restricted actions.We end by noting that the different mechanisms described above for AURKA activation are associated with specific pathologies. Doubly activated AURKA, with bound Tpx2 and Thr288 phosphorylation, is not detected on microtubules in normal cells due to the action of PP6. However, this form of AURKA is readily detected in melanoma cells bearing PP6 mutations, which gives rise to pathological chromosome instability and DNA damage (Hammond et al., 2013). Bora is overexpressed in multiple cancer types, including ovarian cancer, where it plays a pro-oncogenic role (Parrilla et al., 2020), and there is significant evidence for ROS signaling, which is involved in centrosomal AURKA activation by Cep192, contributing to a number of disease states. Finally, AURKA itself is overexpressed in numerous cancers associated with drug resistance and poor patient outcome. However, the clinical utility of AURKA inhibitors to date has been limited, likely because of essential roles of AURKA in multiple events in the cell cycle. We posit that the discovery that AURKA is activated through a variety of mechanisms to execute distinct events may afford opportunities to develop drugs targeting a subset of the biological functions of AURKA and hence enable more precise tuning of the therapeutic window.     

When similar beginnings lead to different ends: Constraints and diversity in cirripede larval development

Summary

Cirripedes are fascinating models for studying both functional constraints and diversity in larval development. Adult cirripedes display an amazing variation in morphology from sessile suspension feeders that still retain many crustacean characters to parasites that have lost virtually all arthropod traits. In contrast, cirripede larval development follows a common scheme with pelagic larvae comprising a series of nauplii followed by a cyprid. Variations are mostly concerned with whether or not the nauplii are feeding and the degree of abbreviation of development, culminating in species where the larvae hatch as cyprids. The cypris larvae are very similar among the ingroups of the Cirripedia, but interesting variations occur in structures used for substrate location and attachment. The cyprid is specialized to both swim through the water and actively explore the substratum by walking on the antennules and using an array of sensory organs in search for a suitable site to attach. This unique morphology and behavior of the cyprid have enabled the Cirripedia to colonize widely different habitats ranging from hard rock to soft animal tissue. Yet, the cyprid can metamorphose into juveniles as different as a setose feeding barnacle and the vermiform stages of the parasitic forms. This emphasizes the importance of the cyprid as one of the key features for the evolutionary success of the Cirripedia.     

Modulation of developmental signals by endocytosis: different means and many ends

Recent advances have highlighted the importance of endocytic processes in regulating the activity and distribution of developmental signals. Classically, signalling is downregulated by endocytosis and subsequent trafficking to lysosomes (e.g. Notch, Hedgehog, Roundabout). However, endocytosis can also have a positive role in signalling. For example, endocytosis of Delta, the ligand of Notch, is needed for activation of the signal. In the case of signalling by Hedgehog, endocytic trafficking segregates an inhibitory receptor (Patched) from the positive effector (Smoothened). Endosomes could also be the site where signalling is activated (e.g. transforming growth factor beta). Finally, endocytosis could power the transport of morphogens along epithelia.     

Roles of the Kinase TAK1 in TRAF6-Dependent Signaling by CD40 and Its Oncogenic Viral Mimic, LMP1

The Epstein-Barr virus (EBV)-encoded protein latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE). LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR) superfamily member CD40, and relies on TNFR-associated factor (TRAF) adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6) production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals.     

Comparative analysis of signal transduction by CD40 and the Epstein-Barr virus oncoprotein LMP1 in vivo

There is much evidence, based primarily on in vitro studies, indicating that the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1) mimics an activated CD40 receptor. In order to investigate the extent of similarity between LMP1 and CD40 functions in vivo, we analyzed the cytoplasmic signaling properties of LMP1 and CD40 in B cells in a directly comparable manner. For this purpose, we generated transgenic mice expressing either LMP1 or a chimeric LMP1CD40 molecule, which constitutively activates the CD40 pathway, under the control of the CD19 promoter. LMP1 and LMP1CD40 were expressed at similar levels in a B-lymphocyte-specific manner. Similar to LMP1, LMP1CD40 suppressed germinal center (GC) formation and antibody production in response to thymus-dependent antigens, albeit to a greater extent than LMP1. Furthermore, the avidity of the antibodies produced against thymus-dependent antigens was lower for LMP1CD40 transgenic mice than for wild-type and LMP1 transgenic mice. GC suppression was linked to the ability of LMP1CD40 and LMP1 to downregulate mRNA and protein levels of BCL6 and to suppress the activity of the BCL6 promoter. In contrast to LMP1, LMP1CD40 caused an upregulation of CD69, CD80, and CD86 in B cells and a dramatic increase in serum immunoglobulin M. In addition, LMP1CD40 but not LMP1 transgenic mice had elevated numbers of marginal-zone B cells and increased populations of polymorphonuclear cells and/or neutrophils. Consistent with these findings, LMP1CD40 but not LMP1 transgenic mice showed signs of spontaneous inflammatory reactions and the potential for autoimmunity.     

The viral oncoprotein LMP1 exploits TRADD for signaling by masking its apoptotic activity

The tumor necrosis factor (TNF)-receptor 1–associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-κB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-κB kinase β (IKKβ). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1′s TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-κB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function.     

LMP1 protein from the Epstein-Barr virus is a structural CD40 decoy in B lymphocytes for binding to TRAF3

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.     

Evaluation of CD40, its ligand CD40L and Bcl-2 in psoriatic patients

Psoriasis is a chronic, recurrent, inflammatory disease. Recent investigations indicate an autoimmune pathogenesis of the disease. Apoptosis plays an important role in the regulation of immune mechanisms in many autoimmune diseases. Although CD40, CD40L, and Bcl-2 have already been studied in psoriatic skin lesions, little is known about their circulating forms. The aim of the present study was to evaluate the serum concentrations of Bcl-2, soluble CD40 and CD40L in psoriatic patients. The study was performed using ELISA kits in 39 psoriatic patients before treatment and after two weeks of topical ointment. Data was analyzed with respect to severity of psoriasis, duration of the disease, and coexisting psoriatic arthritis. Our results revealed that serum concentrations of soluble CD40 and CD40L before and after treatment were significantly higher (p < 0.01 and p < 0.001) in patients with psoriasis compared to the control group. Topical treatment of psoriatic lesions with dithranol ointment failed to decrease serum of CD40 and CD40L, which has not been described until now. There was no significant difference in serum Bcl-2 concentration between the compared groups. We did not find significant differences in serum concentrations of Bcl-2, CD40 or CD40L between patients with mild or severe psoriasis, nor any correlation between disease duration and the presence of psoriatic arthritis symptoms. Our data indicates upregulation of the CD40/CD40L system in psoriatic patients despite topical treatment and suggests their possible role in the pathogenesis of psoriasis.     

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8⁺ and CD4⁺ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8⁺ and CD4⁺ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4⁺ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4⁺ T cells was significantly correlated with that of CD8⁺ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4⁺ T cell responses showed more significant changes than CD8⁺ T cell responses. CD8⁺ and CD4⁺ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4⁺ T cells secreted significantly higher cytokine levels than did CD8⁺ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.     

Diagnostic utility of APOE, soluble CD40, CD40L, and Abeta1-40 levels in plasma in Alzheimer's disease

A continuous inflammatory state is associated with Alzheimer's disease (AD) evidenced by an increase in proinflammatory cytokines around beta-amyloid (Abeta) deposits. In addition, functional loss of CD40L is shown to result in diminished Amyloid precursor proton (APP) processing and microglial activation, supporting a prominent role of CD40-CD40L in AD etiology. We therefore hypothesize that a peripheral increase in Abeta may result in corresponding increase of sCD40 and sCD40L further contributing to AD pathogenesis. We measured plasma Abeta, sCD40 and sCD40L levels in 73 AD patients and compared to 102 controls matched on general demographics. We demonstrated that Abeta(1-40), levels of sCD40 and sCD40L are increased in AD and declining MMSE scores correlated with increasing sCD40L, which in turn, correlated positively with Abeta(1-42). We then combined sCD40, sCD40L, Abeta and APOE and found that this biomarker panel has high sensitivity and specificity (>90%) as a predictor of clinical AD diagnosis. Given the imminent availability of potentially disease modifying therapies for AD, a great need exists for peripheral diagnostic markers of AD. Thus, we present preliminary evidence for potential usefulness for combination of plasma sCD40, sCD40L along with Abeta(1-40) and APOE epsilon4 in improving the clinical diagnosis of AD.     

LMP1, a viral relative of the TNF receptor family,signals principally from intracellular compartments

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded, ligand-independent receptor that mimics CD40. We report here that LMP1 signals principally from intracellular compartments. LMP1 associates simultaneously with lipid rafts and with its signaling molecules, tumor necrosis factor-receptor (TNF-R)-associated factors (TRAFs) and TNF-R1-associated death domain protein (TRADD) intracellularly, although it can be detected at low levels at the plasma membrane, indicating that most of LMP1's signaling complex resides in intracellular compartments. LMP1's signaling is independent of its accumulation at the plasma membrane in different cells, and as demonstrated by a mutant of LMP1 which has significantly reduced localization at the plasma membrane yet signals as efficiently as does wild-type LMP1. The fusion of the transmembrane domain of LMP1 to signaling domains of CD40, TNF-R1 and Fas activates their signaling; we demonstrate that a fusion of LMP1 with CD40 recruits TRAF2 intracellularly. Our results imply that members of the TNF-R family can signal from intracellular compartments containing lipid rafts and may do so when they act in autocrine loops.     

The viral mimic, polyinosinic:polycytidylic acid, induces fever in rats via an interleukin-1-dependent mechanism

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded RNA that is used experimentally to model viral infections in vivo. Previous studies investigating the inflammatory properties of this agent in rodents demonstrated that it is a potent pyrogen. However, the mechanisms underlying this response have not been fully elucidated. In the current study, we examined the effects of peripheral administration of poly I:C on body temperature and cytokine production. Male rats were implanted with biotelemetry devices and randomly assigned to one of the following three groups: poly I:C + saline, poly I:C + interleukin-1 receptor antagonist (IL-1ra), or saline + saline. Maximal fever of 1.6 degrees C above baseline was observed 3 h after an intraperitoneal injection of poly I:C (750 microg/kg). Pretreatment with IL-1ra diminished this response by >50% (maximum body temperature = 0.6 degrees C above baseline). Plasma IL-6 concentration increased fivefold 2 h post-poly I:C compared with saline-injected rats; levels returned to baseline 4 h postinjection. Pretreatment with IL-1ra prevented this rise in IL-6. Plasma tumor necrosis factor (TNF)-alpha was also increased more than fourfold 2 h postinjection but remained unaffected by IL-1ra treatment. IL-1beta and cyclooxygenase-2 mRNA were significantly upregulated in the hypothalamus of poly I:C-treated animals. Finally, poly I:C decreased food intake by 30%, but this response was not altered by pretreatment with IL-1ra. These results suggest that poly I:C induces fever, but not anorexia, through an IL-1 and prostaglandin-dependent mechanism.     

CD20, CD3, and CD40 ligand microclusters segregate three-dimensionally in vivo at B-cell-T-cell immunological synapses after viral immunity in primate brain

The clearance of virally infected cells from the brain is mediated by T cells that engage antigen-presenting cells to form supramolecular activation clusters at the immunological synapse. However, after clearance, the T cells persist at the infection site and remain activated locally. In the present work the long-term interactions of immune cells in brains of monkeys were imaged in situ 9 months after the viral inoculation. After viral immunity, the persistent infiltration of T cells and B cells was observed at the infection sites. T cells showed evidence of T-cell receptor signaling as a result of contacts with B cells. Three-dimensional analysis of B-cell-T-cell synapses showed clusters of CD3 in T cells and the segregation of CD20 in B cells, involving the recruitment of CD40 ligand at the interface. These results demonstrate that immunological synapses between B cells and T cells forming three-dimensional microclusters occur in vivo in the central nervous system and suggest that these interactions may be involved in the lymphocyte activation after viral immunity at the original infection site.