IgE receptor of mast cells signals mediator release and inflammation via adaptor protein 14-3-3ζ.

BACKGROUND: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation-dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. OBJECTIVE: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. METHODS: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. RESULTS: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. CONCLUSION: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr-binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.

Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order, and molecular chaperone activity.

The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer-monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein.

Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.

14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.

A selective ATP-competitive sphingosine kinase inhibitor demonstrates anti-cancer properties.

The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.

Sphingosine and FTY720 directly bind pro-survival 14-3-3 proteins to regulate their function.

The dimeric 14-3-3 protein family protects cells from apoptosis by regulating pro-apoptotic molecules. Conversely, the cationic lipid sphingosine is associated with physiological apoptosis and induces apoptosis in its own right by a largely undefined mechanism. We show here that sphingosine and 14-3-3 interact directly in the control of cell death. The binding of sphingosine to 14-3-3 proteins renders them phosphorylatable at the dimer interface, an event that abolishes the pro-survival signalling of 14-3-3. Sphingosine kinase 1 reduces availability of sphingosine for interaction with 14-3-3, thus inhibiting cell death and providing a new mechanistic insight into the role of this enzyme in cell survival and oncogenesis. Importantly, FTY720, a sphingosine analogue with apoptotic activity that is currently in phase III clinical trials for multiple sclerosis, acts in a similar manner to sphingosine in potentiating 14-3-3 phosphorylation. The biological significance of 14-3-3 phosphorylation was demonstrated with a non-phosphorylatable 14-3-3zeta mutant which retarded apoptosis induced by sphingosine and FTY720. These results demonstrate that direct association of sphingosine with 14-3-3 is required for 14-3-3 phosphorylation, and that this axis can control cell fate. Furthermore, these results suggest a new therapeutic activity for FTY720 as an anti-cancer agent based on this mechanism.

Human immunodeficiency virus 1 (HIV-1) virion infectivity factor (Vif) is part of reverse transcription complexes and acts as an accessory factor for reverse transcription.

Virion infectivity factor (Vif) facilitates HIV infection by counteracting APOBEC3G late in replication in virus-producer cells. Here, we show that early after infection of new target cells Vif is part of the HIV reverse transcription machinery and acts as an accessory factor for reverse transcription. Vif protein was present in gradient fractions containing reverse transcription complexes (RTCs), and anti-Vif antibody immunoprecipitated HIV reverse transcription products from these gradient fractions. To investigate a role for Vif in RTCs independent of APOBEC3G, we created an intracellular environment that would restrict reverse transcription by pre-treating permissive target cells with 5-Fluoro 2-deoxyuridine, a thymidylate synthetase inhibitor, prior to infection with virus from permissive cells. Infectivity assays and quantitation of reverse transcription products demonstrated that replication of HIV lacking Vif was inhibited to a greater degree than wild type, without concurrent mutation of reverse transcription products, suggesting compromised reverse transcription in the absence of Vif.