The full-length interleukin-33 (FLIL33)–importin-5 interaction does not regulate nuclear localization of FLIL33 but controls its intracellular degradation

Andrew Clerman,Zahid Noor,Rita Fishelevich,Virginia Lockatell,Brian S Hampton,Nirav G Shah,Mariah V Salcedo,Nevins W Todd,Sergei P Atamas,Irina G Luzina

doi:10.1074/jbc.m117.807636

Abstract

Human mature IL-33 is a member of the IL-1 family and a potent regulator of immunity through its pro-T helper cell 2 activity. Its precursor form, full-length interleukin-33 (FLIL33), is an intranuclear protein in many cell types, including fibroblasts, and its intracellular levels can change in response to stimuli. However, the mechanisms controlling the nuclear localization of FLIL33 or its stability in cells are not understood. Here, we identified importin-5 (IPO5), a member of the importin family of nuclear transport proteins, as an intracellular binding partner of FLIL33. By overexpressing various FLIL33 protein segments and variants in primary human lung fibroblasts and HEK293T cells, we show that FLIL33, but not mature interleukin-33, physically interacts with IPO5 and that this interaction localizes to a cluster of charged amino acids (positions 46-56) but not to an adjacent segment (positions 61-67) in the FLIL33 N-terminal region. siRNA-mediated IPO5 knockdown in cell culture did not affect nuclear localization of FLIL33. However, the IPO5 knockdown significantly decreased the intracellular levels of overexpressed FLIL33, reversed by treatment with the 20S proteasome inhibitor bortezomib. Furthermore, FLIL33 variants deficient in IPO5 binding remained intranuclear and exhibited decreased levels, which were also restored by the bortezomib treatment. These results indicate that the interaction between FLIL33 and IPO5 is localized to a specific segment of the FLIL33 protein, is not required for nuclear localization of FLIL33, and protects FLIL33 from proteasome-dependent degradation.

Highlights

Human mature IL-33 is a member of the IL-1 family and a potent regulator of immunity through its pro-T helper cell 2 activity
By overexpressing various full-length interleukin-33 (FLIL33) protein segments and variants in primary human lung fibroblasts and HEK293T cells, we show that FLIL33, but not mature interleukin-33, physically interacts with IPO5 and that this interaction localizes to a cluster of charged amino acids but not to an adjacent segment in the FLIL33 N-terminal region. siRNA-mediated IPO5 knockdown in cell culture did not affect nuclear localization of FLIL33
These results indicate that the interaction between FLIL33 and IPO5 is localized to a specific segment of the FLIL33 protein, is not required for nuclear localization of FLIL33, and protects FLIL33 from proteasome-dependent degradation

Summary

To whom correspondence should be addressed

Receptor-mediated cytokine activity of IL-33 is driven by its C-terminal region, termed mature IL-33 (MIL33), which is released as a proteolytic product of the precursor, or full-length IL-33 (FLIL33). Alternatively spliced variants of IL-33 lacking segments of the N terminus were primarily localized to the cytoplasm, secreted extracellularly, and retained the capacity for receptor binding and immunologic effects. Increased levels of these transcripts were found in the airways of asthma patients with type 2 inflammation [20]. We have recently reported that intracellular levels of FLIL33 protein are regulated by Th1 and Th2 cytokines post-translationally through proteasomal degradation [21]. We show that the interaction between IPO5 and FLIL33 results in stabilization of the intracellular FLIL33 protein by protecting it from proteasome-dependent degradation

Results

Discussion

Experimental procedures