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A5741 - Importin-5 Controls the Intracellular Degradation of IL-33 Precursor in Primary Pulmonary Fibroblasts
Author Block: A. Clerman, N. G. Shah, N. W. Todd, S. P. Atamas, I. G. Luzina; University of Maryland School of Medicine, Baltimore, MD, United States.
RATIONALE: Interleukin (IL)-33 regulates pulmonary pathobiology in its two biologically distinct forms, mature IL-33 (MIL33) and precursor, or full-length (FLIL33). MIL33 acts as a potent mediator of Th2 responses through the T1/ST2 receptor, whereas FLIL33 is basally expressed in the nucleus of many cell types, and is thought to regulate inflammation. Elevated levels of IL-33 have been shown in asthma, COPD, and pulmonary fibrosis. We have previously reported that intracellular levels of FLIL33 are regulated by its proteasomal degradation. The precise molecular mechanisms controlling nuclear localization and the intracellular stability of FLIL33 need to be elucidated.
METHODS: Recombinant plasmid or adenoviral constructs encoding FLIL33, its various N-terminal segment mutants, MIL33, or FLIL37, all HA-tagged on the C-terminus were prepared, validated, and used for gene delivery to primary normal human lung fibroblast (NHLF) cultures from adult donors or HEK293T cells. LC-MS/MS of co-immunoprecipitates were used to identify binding partners of FLIL33. Western blotting was used to assess protein levels. Subcellular localization was assessed by immunocytochemistry and western blotting of cytoplasmic and nuclear cell fractions. RNA interference was used to attenuate protein expression levels.
RESULTS: Importin-5 (IPO5) consistently co-immunoprecipitated with FLIL33, but not MIL33 or FLIL37. This interaction occurred in NHLFs, HEK293T cells, and in an in vitro pull-down assay using purified FLIL33 and IPO5. Immunoprecipitation of N-terminal FLIL33 mutants further localized the interaction to the region between amino acids (aa) 45-67. Poly-alanine substitution or deletion mutants of aa46-56, but not of aa61-67, abolished IPO5 co-immunoprecipitation. Nuclear localization of FLIL33 was unchaged by IPO5 knockdown with siRNA or the aa45-56 mutation. IPO5 knockdown with siRNA, as well as substitution or deletion mutations of aa45-56, but not of aa61-67 used as a control, resulted in markedly reduced levels of FLIL33 protein in NHLFs. These reductions were reversed by inhibition of the 20S proteasome with bortezomib.
CONCLUSION: We report an interaction between FLIL33 and importin-5 (IPO5), a karyopherin that transports target proteins into the nucleus. Surprisingly, this interaction does not appear to control the nuclear localization of FLIL33. Rather, it protects FLIL33 from proteasome-dependent degradation. FLIL33 binds IPO5 through its N-terminus and requires aa45-56. Considering that IL-33 exists as a precursor that is basally expressed and is elevated in a number of pulmonary disease states, better understanding of its intracellular activity and mechanisms that maintain the intracellular pool could lead to the development of innovative therapeutic strategies.