Inflammation and fibrosis often co-occur and contribute to a remarkably broad variety of diseases of every organ and tissue. The overall role of interleukin (IL)-33 in inflammation and fibrosis has already been established, but the mechanisms of such regulation are not fully understood. Limited attention has been paid to the IL-33 precursor?full-length IL-33 (FLIL33)?which is basally and inducibly expressed, resides mostly in the cell nucleus, and is thought to regulate inflammatory responses, wound healing, and transcriptional regulation independently of the mature IL-33 (MIL33) cytokine. The understudied FLIL33 requires more attention, because it is both the immediate source of the MIL33 cytokine and an independently active factor. These IL-33 forms become pathophysiologically engaged under stress, but IL-33-null mice have no noticeable basal phenotype, suggesting that IL-33 depletion is a safe therapeutic approach. Our objective is to form the basis for therapeutic manipulation of IL-33 in inflammatory fibrotic diseases through integrated understanding of proteolytic maturation and extracellular release of MIL33, intracellular signaling and functioning of FLIL33, and proteolytic stability of the FLIL33 protein pool. We amassed new data related to the molecular control of IL-33 subcellular localization, functional maturation, and extracellular release through a previously unknown region within the FLIL33 molecule, which spans substantially more of the N-terminus than the currently known ?sensor domain.? We hypothesize that this segment may be targeted to control IL-33 activation and extracellular release. We also discovered that the intracellular function of FLIL33 is centered on Smad3 phosphorylation in a TGF-beta-independent fashion. We hypothesize that this process is mediated by the adaptor-related protein complex 2 and that targeting this mechanism allows for abrogation of the functional effects of intracellular FLIL33. We also recently reported that importin 5 (IPO5) protects FLIL33 from proteolytic degradation, driving the hypothesis that IPO5-binding, cell-permeable decoy peptide(s) will induce the loss of IPO5-mediated protection of FLIL33 from proteasomal degradation. This prospective therapy will deplete the FLIL33 protein pool, thus exhausting the source of MIL33 and simultaneously attenuating the intracellular effects ofFLIL33.
The Specific Aims of this project are to: 1) Precisely map the segments in the N-terminal region of FLIL33 that are responsible for its nuclear-versus-cytoplasmic localization, functional maturation, extracellular release, and selective binding of intracellular molecular partners; 2) Define the molecular mechanism responsible for FLIL33-induced, TGF-?ligand-independent phosphorylation of Smad3; and 3) Develop a cell-permeable decoy peptide-based approach to deplete intracellular FLIL33, thereby exhausting the source of MIL33 and simultaneously attenuating the independent effects of FLIL33. Evaluate the in vivo efficacy of such FLIL33 depletion. Achieving these goals will offer new understanding of IL-33 pathobiology and initiate the development of precise, innovative therapeutic modalities.
In many diseases that are difficult to treat, body tissues and whole organs become inflamed and accumulate excess scar tissue in a process call fibrosis. The mechanisms through which cells and molecules become disturbed in these diseases are complex and incompletely understood, delaying the development of new medicines. In this project, we explore previously understudied aspects of a body molecule called IL-33, which may be targeted with future therapies, and begin developing one of such innovative therapies.
