ROLE OF INTRACELLULAR MEMBRANE SIGNALOSOMES IN B CELL NF-KAPPAB ACTIVATION

Role of intracellular membrane signalosomes in B cell NF-kB activationThis proposal focuses on a novel mechanism that underlies NF-kB activation in peripheral B cell differentiationand, when dysregulated in autoreactive B cells, promotes pathogenic autoantibody responses in autoimmunediseases, such as lupus. As we contend, this mechanism is underpinned by the formation of intracellularmembrane signalosomes (IM signalosomes), e.g., as mediated by the interaction of signal adaptor TRAF6, anE3 ubiquitin ligase, with Rab7, an endosome-tethered small GTPase, in B cells upon CD40 engagement. Ourcontention, if successfully tested, would lend a strong support to the emerging paradigm that IM structures relaysignals to specify cellular processes. It would also identify a new target of therapeutics with less side effects, asdisrupting IM signalosomes will not affect signals important for homeostasis. In B cells stimulated with a CD40 or TLR ligand, TRAF6 plays an important role in NF-kB activation, whichis required for AID induction and, therefore, Ig class switch DNA recombination (CSR) and antibody responses.As shown by our IM fractionation of B cells stimulated with CD154 (CD40 ligand), TRAF6 was ? unexpectedly ?localized mainly in mature endosomes, to which Rab7 was tethered. This, together with direct interaction andco-immunoprecipitation of TRAF6 with Rab7, as well as CD40 internalization and co-localization with Rab7,prompted us to hypothesize that IM signalosomes form on endosomes (through multi-valent CD40/TRAF6/Rab7interactions) to mediate sustained NF-kB activation. This is supported by our studies using pharmacologicalinhibition and genetic ablation approaches, as CD154- and TLR-induced AID/CSR was hampered by a smallmolecule inhibitor of endocytosis (dynasore) or Rab7 (CID 106700), or Rab7 gene knockout, and CID 1067700-treated mice and B cell-specific Rab7 knockout (KO) mice show severely impaired antibody responses while Bcell homeostasis and functions of other immune cells are normal. Finally, lupus B cells displayed moreCD40 internalization and high Rab7 expression, consistent with our contention that IM signalosomes aredysregulated in these cells to promote NF-kB hyperactivation. To test our hypotheses, we will analyze CD40/TRAF6/Rab7 interactions and TRAF6 K63 polyubiquitinationon endosomes in normal B cells upon stimulation, and verify the block of these processes in C57/Rab7-KO-tdTomato B cells (Aim 1.1). We will further analyze CD40 internalization, high Rab7 expression and NF-kBhyperactivation in the same lupus B cells in humans and mice and characterize the human lupus B cell subsetshowing the highest Rab7 expression (Aim 1.2). Finally, we will blunt NF-kB activation by disrupting IMsignalosomes in vitro and in vivo, through generation of a Rab7 mutant that cannot bind TRAF6 (Aim 2.1) andscreening of small molecules to identify those inhibiting the TRAF6/Rab7 interaction (Aim 2.2), therebyhampering CSR and antibody/autoantibody responses. Successful completion of these experiments will bridgea significant gap of our understanding of signal transduction in an important pathophysiological context.