Role of intracellular membrane signalosomes in B cell NF-kappaB activation

University Of Texas Hlth Science Center, San Antonio TX

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Abstract

PROJECT SUMMARY: Role of intracellular membrane signalosomes in B cell NF-kB activation This proposal focuses on a novel mechanism that underlies NF-kB activation in peripheral B cell differentiation and, when dysregulated in autoreactive B cells, promotes pathogenic autoantibody responses in autoimmune diseases, such as lupus. As we contend, this mechanism is underpinned by the formation of intracellular membrane signalosomes (IM signalosomes), e.g., as mediated by the interaction of signal adaptor TRAF6, an E3 ubiquitin ligase, with Rab7, an endosome-tethered small GTPase, in B cells upon CD40 engagement. Our contention, if successfully tested, would lend a strong support to the emerging paradigm that IM structures relay signals to specify cellular processes. It would also identify a new target of therapeutics with less side effects, as disrupting 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, which is 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 and co-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/Rab7 interactions) to mediate sustained NF-kB activation. This is supported by our studies using pharmacological inhibition and genetic ablation approaches, as CD154- and TLR-induced AID/CSR was hampered by a small molecule 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 B cell homeostasis and functions of other immune cells are normal. Finally, lupus B cells displayed more CD40 internalization and high Rab7 expression, consistent with our contention that IM signalosomes are dysregulated in these cells to promote NF-kB hyperactivation. To test our hypotheses, we will analyze CD40/TRAF6/Rab7 interactions and TRAF6 K63 polyubiquitination on 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-kB hyperactivation in the same lupus B cells in humans and mice and characterize the human lupus B cell subset showing the highest Rab7 expression (Aim 1.2). Finally, we will blunt NF-kB activation by disrupting IM signalosomes in vitro and in vivo, through generation of a Rab7 mutant that cannot bind TRAF6 (Aim 2.1) and screening of small molecules to identify those inhibiting the TRAF6/Rab7 interaction (Aim 2.2), thereby hampering CSR and antibody/autoantibody responses. Successful completion of these experiments will bridge a significant gap of our understanding of signal transduction in an important pathophysiological context.